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WO1993011778A1 - Molecules anti-thrombotiques bifonctionnelles et polypetptides anti-trhombotiques - Google Patents

Molecules anti-thrombotiques bifonctionnelles et polypetptides anti-trhombotiques Download PDF

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Publication number
WO1993011778A1
WO1993011778A1 PCT/US1992/010947 US9210947W WO9311778A1 WO 1993011778 A1 WO1993011778 A1 WO 1993011778A1 US 9210947 W US9210947 W US 9210947W WO 9311778 A1 WO9311778 A1 WO 9311778A1
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Prior art keywords
binding
fragment
vwf
polypeptide
platelet
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PCT/US1992/010947
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English (en)
Inventor
Zaverio M. Ruggeri
Jerry L. Ware
Luigi De Marco
Mario Mazzucato
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The Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F02—COMBUSTION ENGINES; HOT-GAS OR COMBUSTION-PRODUCT ENGINE PLANTS
    • F02B—INTERNAL-COMBUSTION PISTON ENGINES; COMBUSTION ENGINES IN GENERAL
    • F02B75/00—Other engines
    • F02B75/02—Engines characterised by their cycles, e.g. six-stroke
    • F02B2075/022—Engines characterised by their cycles, e.g. six-stroke having less than six strokes per cycle
    • F02B2075/027—Engines characterised by their cycles, e.g. six-stroke having less than six strokes per cycle four

Definitions

  • This invention relates to a bifunctional molecule and to •a polypeptide, each of which is useful in the treatment of vascular disorders such as thrombosis.
  • This invention further relates to the production by recombinant DNA-directed methods of pharmacologically useful quantities of the polypeptides of the present invention.
  • hemostasis refers to those processes which comprise the defense mechanisms of the body against loss of circulating blood caused by vascular injury. Processes which are normal as a physiologic response to vascular injury may lead in pathologic circumstances, such as in a patient afflicted with atherosclerotic vascular disease or chronic congestive heart failure, to the formation of undesired thrombi (clots) with resultant vascular occlusion.
  • Impairment of blood flow to organs under such circumstances may lead to severe pathologic states, including myocardial infarction, a leading cause of mortality in developed countries.
  • the restriction or termination of the flow of blood within the circulatory system in response to a wound or as a result of a vascular disease state involves a complex series of reactions which can be divided into two processes, primary and secondary hemostasis.
  • Primary hemostasis refers to the process of platelet plug or soft clot formation.
  • the platelets are non-nucleated discoid structures approximately 2-5 microns in diameter derived from megakaryocytic cells.
  • Effective primary hemostasis is accomplished by platelet adhesion, the interaction of platelets with the surface of damaged vascular endothelium on which are exposed underlying collagen fibers and/or other adhesive macromolecules such as proteoglycans and glycosaminoglycans to which platelets bind.
  • Secondary hemostasis involves the reinforcement or crosslinking of the soft platelet clot. This secondary process is initiated by proteins circulating in the plasma (coagulation factors) which are activated during primary hemostasis, either in response to a wound or a vascular disease state. The activation of these factors results ultimately in the production of a polymeric matrix of the protein fibrinogen (then called fibrin) which reinforces the soft clot. The conversion of fibrinogen to fibrin is catalyzed by thrombin, .one of the coagulation factors. As described below, thrombin also participates in the reactions which activate platelets. Therapeutic drugs for controlling thrombosis have been classified according to the stage of hemostasis which is affected by the administration thereof.
  • anticoagulant therapeutics typically represent a class of drugs which intervene in secondary hemostasis.
  • Anticoagulants typically have no direct effect on an established thrombus, nor do they reverse tissue damage.
  • hazard of hemorrhage Associated with the use of existing anticoagulants is the hazard of hemorrhage, which may under some conditions be greater than the clinical benefits otherwise provided by the use thereof. As a result, anticoagulant therapy must be closely monitored.
  • Certain anticoagulants act by inhibiting the synthesis of vitamin K-dependent coagulation factors resulting in the sequential depression of, for example, factors II, VII, IX, and X.
  • anticoagulants which are used clinically include coumarin, dicoumarol, phenindione, and phenprocoumon.
  • Thrombolytics act by lysing thrombi after they have been formed.
  • Thrombolytics such as streptokinase and urokinase have been indicated for the management of acute myocardial infarctions and have been used successfully to remove intravascular clots if administered soon after thrombosis occurs.
  • the lysis effected thereby may be incomplete and nonspecific, i.e., useful plasma fibrinogen, in addition to fibrin polymers within clots, is affected.
  • a common adverse reaction associated with the use of such therapeutics is hemorrhage.
  • a third classification, antiplatelet drugs includes drugs which suppress primary hemostasis by altering platelets or their interaction with other circulatory system components. The present invention relates to this classification of antiplatelet drugs.
  • Specific antiplatelet drugs operate by one or several mechanisms.
  • a first example involves reducing the availability of ionized calcium within the platelet cytoplasm thereby impairing activation of the platelet and resultant aggregation.
  • Pharmaceuticals representative of this strategy include prostacyclin, and also Persantine ® (dipyridamole) which may affect calcium concentrations by affecting the concentration of cyclic AMP. Numerous side effects related to the administration of these compounds have been reported.
  • An additional class of antiplatelet drugs acts by inhibiting the synthesis of thromboxane A 2 within the platelet, reducing the platelet activation response.
  • Non-steroidal anti- inflammatory agents such as ibuprofen, phenylbutazone and napthroxane may produce a similar effect by competitive inhibition of a particular cyclooxygenase enzyme, which catalyzes the synthesis of a precursor of thromboxane A 2 .
  • a similar therapeutic effect may be derived through the administration of aspirin which has been demonstrated to irreversably acetylate a cyclooxygenase enzyme necessary to generate thromboxane A 2 .
  • a third anti-platelet mechanism has involved the platelet membrane so as to interfere with surface receptor function.
  • ticlopidene a large branched polysaccharide, which is believed to impair the interaction of fibrinogen with platelet receptors that are exposed during aggregation.
  • Dextran is contraindicated for patients with a history of renal problems or with cardiac impairment.
  • the therapeutic ticlopidine is stated to inhibit platelet adhesion and aggregation by suppressing the binding of von Willebrand factor and/or fibrinogen to their respective receptors on the platelet surface.
  • ticlopidene possesses insufficient specificity to eliminate the necessity of administering large doses which, in turn, may be associated with clinical side effects.
  • the aforementioned pharmaceuticals are foreign to the body and may cause numerous adverse clinical side effects, there being no way to prevent such compounds from participating in other aspects of a patient's physiology or biochemistry, particularly if high doses are required. It would be desirable to provide for pharmaceuticals having such specificity for certain of the reactions of hemostasis, that they could be administered to patients at low doses, such doses being much less likely to produce adverse effects in patients.
  • the present invention is directed to the provision of a therapeutic molecule having a plurality of antithrombotic functions which are based on natural components of the hemostatic mechanism.
  • this invention provides for an antithrombotic molecule which is capable of inhibiting the adhesion of platelets to damaged or diseased vascular surfaces and which is capable of inhibiting the activation or aggregation of platelets.
  • the antithrombotic molecule of the present invention is polyfunctional in that the molecule includes a plurality of functional groups of a nature such that the functional groups are capable of interfering with a plurality of materials which are normally involved in the body process of clot formation.
  • One aspect of the present invention comprises a bifunctional molecule which has the capability of affecting two processes in the formation of a thrombus, namely (1) the interaction of thrombins with glycoprotein Ib ⁇ receptor of platelets, and (2) interaction of von Willebrand factor with glycoprotein Ib ⁇ receptor of platelets.
  • One aspect of the present invention is the provision of a bifunctional molecule which is capable of inhibiting activation of a platelet and/or adhesion of a platelet to a damaged or diseased vascular surface and which comprises two linked domains, a first domain capable of inhibiting the binding of thrombins to platelet glycoprotein Ib ⁇ and a second domain capable of inhibiting the binding of von Willebrand factor to platelet glycoprotein Ib ⁇ .
  • structures which comprise one or both of the linked domains of the bifunctional molecule of this invention are antibodies or active fragments thereof, polypeptide fragments derived from proteins that participate in hemostasis, and organic analogs patterned on the aforementioned structures and having the binding activity thereof. Accordingly, the present invention provides also a bifunctional molecule having a first domain selected from the group consisting of:
  • A an antibody, or an active fragment thereof, having as its epitope, or as a part thereof, all or part of a thrombin binding site of the platelet provided by glycoprotein Ib ⁇ ;
  • B a polypeptide comprising approximately the amino terminal His 1 to Arg 293 fragment of glycoprotein Ib ⁇ , in derivatized or underivatized form, or one or more subfragments thereof containing all or part of the thrombin-binding site thereof;
  • C a mutant or derivative of a polypeptide of (B) above; and (D) an organic analog patterned on any of (A) to (C) above and having all or part of the binding activity thereof; and a second domain selected from the group consisting of:
  • A an antibody, or an active fragment thereof, having as its epitope, or as a part thereof, all or part of the von Willebrand factor binding site of glycoprotein Ib ⁇ ;
  • B a polypeptide comprising approximately the Arg 441 to Val 733 fragment of mature von Willebrand factor subunit, in derivatized or underivatized form, or one or more subfragments thereof containing all or part of the glycoprotein Ib ⁇ binding site thereof;
  • C a mutant or derivative of a polypeptide of (B) above; and (D) an organic analog patterned on any of (A) to (C) above and having all or part of the binding activity thereof.
  • a particularly preferred embodiment of a bifunctional molecule having antithrombotic activity is represented by a first domain comprising a fragment of LJ-IblO antibody containing all or part of the variable region thereof and a second domain comprising that fragment of mature von Willebrand factor subunit from approximately residue Arg 441 to approximately residue Val 733 thereof, or a subfragment thereof containing residues Leu 694 to Pro 708 .
  • Another aspect of the present invention is based upon the discovery that certain of the tyrosine residues of the amino terminal region of platelet glycoprotein Ib ⁇ as determined from humans are sulfated thereby facilitating, for example, interaction with thrombins.
  • Peptides or polypeptides containing such sulfated tyrosines have utility as antithrombotics. Accordingly, there is provided a polypeptide comprising approximately the amino terminal His 1 to Arg 293 fragment of glycoprotein Ib ⁇ , in derivatized or underivatized form, or one or more subfragments thereof, said polypeptide containing also a sulfate group attached to the phenolic oxygen of one or more tyrosine residues of said polypeptide.
  • the invention also provides for DNA sequences, expression plasmids and recombinant host cells through which may be expressed numerous of the above-described bifunctional molecules and polypeptides.
  • thrombins and/or von Willebrand factor have been detected in cells other than megakaryocytic cells, for example, cells of the vascular endothelium.
  • This invention provides, therefore, additional strategies for antithrombotic therapy.
  • a bifunctional molecule which is capable of inhibiting activation of a cell and which comprises two linked domains, a first. domain ' capable of inhibiting the binding of thrombins to glycoprotein Ib ⁇ of a cell and a second domain capable of inhibiting the binding of von Willebrand factor to glycoprotein Ib ⁇ of a cell.
  • a further additional strategy for antithrombotic therapy comprises a method for inhibiting activation of endothelial cells, or smooth muscle cells, in a patient, said method comprising administering to said patient an effective amount of a therapeutic composition comprising the amino terminal His 1 to Arg 293 fragment of GPIb ⁇ , in derivatized or underivatized form, or one or more subfragments thereof, and a pharmaceutically acceptable carrier.
  • Figure 1 shows the structure of exemplary peptides, derived from glycoprotein Ib ⁇ , that inhibit binding of thrombins to platelets.
  • Figure 2 shows the effect of an unsulfated synthetic peptide, corresponding to residues 265-285 of glycoprotein Ib ⁇ , on the binding of thrombins to platelets.
  • Transcribed Strand a DNA strand whose nucleotide sequence is read 3' ⁇ 5' by RNA polymerase to produce mRNA, being also referred to as the "noncoding strand”.
  • Reducing Conditions refers to the presence of a "reducing" agent in a solution containing von Willebrand factor, or polypeptides derived therefrom, which agent causes the disruption of disulfide bonds of the vWF.
  • Viral Expression Vector a vector similar to an expression plasmid except that the DNA may be packaged into a viral particle that can transfect cells through a natural biological process.
  • Downstream - a nucleotide of a transcribed strand of a structural gene is said to be downstream from another section of the gene if the nucleotide is normally read by RNA polymerase after the earlier section of the gene; the complimentary nucleotide of the nontranscribed strand, or the corresponding base pair within the double stranded form of the DNA, are also denominated downstream.
  • a restriction endonuclease sequence added upstream (or 5') to the gene means it is added before the sequence encoding the amino terminal end of the protein, while a modification created downstream (or 3') to the structural gene means that it is beyond the carboxy terminus-encoding region thereof.
  • Derivatized Polypeptide a polypeptide which comprises an amino acid sequence that contains also additional groups, such as, but not limited to, sulfate, esters, additional amino acid sequence, glycosyl groups, or glycosylation as may be added to the polypeptide by posttranslational modification in cells, or as may be added artificially;
  • underivatized polypeptide is one consisting solely of an amino acid sequence
  • GPIb ⁇ and vWF as produced in mammalian cells, are typically glycosylated and therefore derivatized, but underivatized forms thereof are also useful in the practice of the invention.
  • Thrombins those forms of thrombin which, at physiologically relevant concentrations, bind to glycoprotein Ib ⁇ of platelets or cells, such binding (interaction) causing in whole or part, directly or indirectly, platelet (or cell) activation;
  • ⁇ -thrombin is such a form of thrombin whereas y- thrombin is not;
  • ⁇ -, e- and f-thrombins as those molecules are known in the art, are examples of thrombins within the definition used herein.
  • thrombin exists in many other forms, in such forms being within the definition herein if their behavior is consistent with the above-identified functional criteria.
  • Biological Activity one or more functions, effects of, activities performed or caused by a molecule in a biological 5 context (that is, in an organism or in an in vitro facsimile) ;
  • a characteristic biological activity of the 52/48 kba monomeric fragment of the mature von Willebrand factor subunit is the potential ability to bind to only one platelet GPIb ⁇ receptor thereby enabling the molecule to inhibit modulator (such as botrocetin) or stimulus-induced binding of multimeric vWF to platelets;
  • modulator such as botrocetin
  • other resultant or related effects of the undimerized 52/48 kDa species include inhibition of platelet activation, aggregation, and also adhesion to surfaces, and the inhibition of thrombosis;
  • a characteristic biological activity of "thrombins" is binding to high affinity sites on platelets in glycoprotein Ib ⁇ , said binding being associated with platelet activation; an additional activity of thrombins is acting as an enzyme to convert fibrinogen into fibrin
  • bifunctional molecules and polypeptides of the invention With respect to the provision of the bifunctional molecules and polypeptides of the invention the following should be noted. It is preferred to use human amino acid and nucleic acid sequences for the production of the bifunctional molecules and polypeptides. However, it is within the scope of the invention to so utilize the sequences of other mammals, in particular where the amino acid or nucleic acid sequence domain of interest possesses no mutations (in comparison with the human sequence) or possesses mutations, provided that they do not cause adverse effects, for example, initiate an immune response in a patient that cannot be safely controlled when the resultant product is administered in otherwise therapeutic doses.
  • the present invention encompasses also the use of bifunctional molecules or polypeptides (and/or the encoding human DNA sequences therefor) that contain, relative to the normal human sequence, mutations which do not affect significantly the biological activity thereof.
  • bifunctional molecules or polypeptides and/or the encoding human DNA sequences therefor
  • the designs of the bifunctional molecules and of the polypeptides of the present invention are based upon the interaction of each of the proteins von Willebrand factor and thrombins with glycoprotein Ib ⁇ receptors of platelets or cells.
  • von Willebrand factor and thrombins
  • glycoprotein Ib ⁇ receptors of platelets or cells For background purposes, there is set forth hereafter information concerning these macromolecules and their role in hemostasis and thrombosis.
  • Background Information - vWF, GPIb ⁇ and Thrombin von Willebrand factor (“vWF”) performs an essential role in normal hemostasis during vascular injury and is also of central importance in the pathogenesis of acute thrombotic occlusions in diseased blood vessels. Both of these roles involve the interaction of vWF with platelets which are induced to bind at the affected site and are then crosslinked. It is believed that single platelets first adhere to a thrombogenic surface after which they become activated, a process involving major metabolic changes and significant morphological
  • Activation is evidenced by the discharge of platelet storage granules containing adhesive substances such as additional vWF (an adhesive protein) , secretion of calcium ions, and the expression on the surface of the platelet of additional functional adhesive sites.
  • additional vWF an adhesive protein
  • platelet cells become aggregated, a process which involves extensive crosslinking of the platelets with additional types of adhesive proteins.
  • platelets are also activated by thrombin.
  • the adhesion of platelets to damaged or diseased vessels occurs through mechanisms that involve specific platelet membrane receptors which, interact with specialized adhesive molecules.
  • glycoprotein Ib- X comple which consists of a noncovalent association of two integral membrane proteins, glycoprotein lb (GPIb) and glycoprotein IX (GPIX) .
  • GPIb is a two-chain molecule having an apparent molecular mass of approximately 160 kDa.
  • GPIb is composed of a heavy (alpha, or "GPIb ⁇ ") chain, having a molecular mass of approximately 145 kDa, linked by disulfide bonds to a light (beta, or GPIb/3) chain, having a molecular mass of approximately 22 kDa.
  • GPIb is an integral membrane protein and both the alpha- and beta- chains described above have transmembrane domains.
  • the adhesive ligand of the GPIb-IX complex is the protein vWF which is found as a component of the subendothelial matrix, as a component of the ⁇ -granules secreted by activated platelets, and also as a circulating blood plasma protein.
  • the actual binding site of vWF on the GPIb-IX receptor has been localized on the amino terminal (His'-Arg 293 ) region of the ⁇ chain of glycoprotein lb. This region of the polypeptide may be prepared as a fragment of GPIb ⁇ having a molecular weight of 45 kDa using, for example, trypsin to effect the necessary proteolytic cleavage.
  • antithrombotic bifunctional molecules and of the polypeptides of the invention are patterned upon or directed to this amino terminal region (45 kDa) of GPIb ⁇ .
  • Inhibition of vWF-GPIb ⁇ interaction results in the prevention of primary hemostasis and the induction of an anti-thrombotic state useful in prevention of diseases in which occlusion of blood vessels plays an important role.
  • inhibition of thrombin-GPIb ⁇ interaction is similarly important.
  • vWF exists as a series of high molecular weight multimers of up to 30 glycosylated subunits per multimer in which the subunits are believed to be identical, with each having an approximate molecular weight of 270,000 (270 kDa).
  • vWF vascular endothelial growth factor
  • a bridging function in which surface bound multimeric vWF binds on the one side to components of the subendothelium, such as collagen or proteoglycans, and on the other side to the GPIb ⁇ receptor of a platelet membrane.
  • vWF is necessary for thrombus formation has been provided by studies using anti-vWF monoclonal antibodies to induce a deficiency in circulating vWF, Bellinger, D.A. et al., Proc. Natl. Acad. Sci. , USA.
  • vWF glycoprotein Ib-IX complex
  • GPIb( ⁇ ) glycoprotein Ib-IX complex
  • the rapidly accumulating platelets are also crosslinked (aggregated) by the binding of fibrin (produced itself from fibrinogen by the action of thrombin) at platelet glycoprotein Hb-IIIa receptor sites, and also by the binding of vWF at these sites, and/or at additional glycoprotein Ib-IX receptor sites.
  • the glycoprotein lib/Ilia receptor may also be involved in the formation of the initial monolayer of platelets.
  • tetrapeptide Arg-GlyAsp-Ser (SEQ ID NO: 1) (vWF residues 1744 to 1747) , a sequence which vWF shares with many other adhesive proteins, is believed to represent the platelet glycoprotein Hb-IIIa binding site.
  • SEQ ID NO: 1 The tetrapeptide Arg-GlyAsp-Ser (SEQ ID NO: 1) (vWF residues 1744 to 1747) , a sequence which vWF shares with many other adhesive proteins, is believed to represent the platelet glycoprotein Hb-IIIa binding site.
  • the multimeric and multivalent character of circulating vWF which enables the macromolecule to effectively carry out its binding and bridging functions.
  • Thrombin also performs an essential role in normal hemostasis during vascular injury and is also of central importance in the pathogenesis of acute thrombotic occlusions in diseased blood vessels. Certain of these functions involve the binding (interaction) of thrombin with platelet GPIb ⁇ . Interfering with this binding is a central achievement of the present development.
  • thrombin a protein having a molecular weight of about 37,000, is one of the most important components of the hemostasis process, catalyzing or regulating numerous key reactions thereof.
  • the properties of thrombin are reviewed in, for example, Fenton, J.W. et al.. Blood Coagulation and Fibrinolvsis. 2, 69-75 (1991), Mann, K.G. et al. , Annals of the New York Academy of Sciences. 614, 63-75 (1991). Briefly, thrombin is a serine protease, produced from proteolysis of its precursor protein, prothrombin.
  • thrombin most important functions related to hemostasis are: (A) catalyzing the conversion of fibrinogen to fibrin monomers which polymerize as a matrix reinforcing soft platelet clots; and (B) activation of platelets.
  • Thrombin also participates in many other cellular processes, including stimulation of smooth muscle contraction, stimulation of endothelial cells to release tissue plasminogen activator, alteration of the permeability of endothelial cell monolayers, and acting as a itogen toward fibroblasts. Consistent with the complex scope of its functions, thrombin is also known to contain exocites, which are sites in its tertiary structure other than the catalytic site where substrates or inhibitors bind and align for more efficient interaction.
  • thrombins forms of thrombin which are useful in thp practice of the invention.
  • thrombins a further important aspect of the present development is the recognition that the binding domains of GPIb ⁇ for thrombin and for vWF are in close . . proximity, making possible the design of bifunctional antithrombotic molecules. As stated above, these processes are normal as a physiologic response to vascular injury.
  • von Willebrand factor which circulates in the blood exists as a series of high molecular weight multimers containing up to 30 glycosylated subunits per multimer in which the subunits are believed to be identical, each having an approximate molecular weight of about 270 kDa.
  • the circulating "mature" human subunit consists of 2050 amino acid residues. Its structure is the final result of extensive post-translational processing.
  • the domain of the vWF subunit which binds to the platelet membrane glycoprotein Ib-IX receptor (GPIb( ⁇ )) has been identified within a fragment of vWF.
  • the fragment may be generated by trypsin digestion, followed by disulfide reduction, and extends from approximately residue 449
  • the above-mentioned residue 449-728 fragment has a molecular weight of about 50 kilo daltons (50 kDa) .
  • This 50,000 molecular weight fragment is referred to also as a
  • 52/48 fragment reflecting the fact that human enzyme systems glycosylate the fragment contributing to its - molecular weight.
  • the amount of glycosylation varies from molecule to molecule, with two weights, 52,000 and 48,000, being most common. Without the additional weight contributed by glycosylation, the polypeptide has a molecular weight of approximately 38,000. Very little polymorphism has been reported in the 52/48 human sequence with one significant exception - histidine/aspartic acid at position 709, see Mancuso, D.J. et al. J. Biol. Chem. , 264(33), 19514-19527, Table V, (1989) .
  • Polypeptides derived from the above mentioned region of the circulating (mature) von Willebrand factor subunit - from approximately residue 449 to approximately residue 728, or subfragments thereof are considered useful as antithrombotic pharmaceuticals when added to blood in such sufficient amounts as to compete successfully with multimeric vWF for platelet GPlb( ⁇ ) receptor sites, thereby preventing monolayer formation by, or crosslinking of, the platelets in circumstances where thrombus formation is undesirable, such as in the treatment of vascular disorders.
  • a fragment of multimeric von Willebrand factor from the blood having platelet glycoprotein Ib( ⁇ ) binding activity and of approximately 116,000 (116 kDa) molecular weight is isolated by digesting vWF with trypsin. Treatment of the 116 kDa fragment with a reducing agent capable of cleaving disulfide bonds results ir; the formation of a pair of identical fragments. Each of the identical fragments (which together comprise the 116 kDa polypeptide) has an apparent molecular weight of about 50 kDa. (Polypeptide molecular weight are typically measured by migration, relative to standards, in a denaturing gel electrophoresis system.
  • the 52/48 fragment has been demonstrated to competitively inhibit the binding of vWF to platelets.
  • manipulation of the 52/48 fragment or its unglycosylated 38 kDa equivalent has proved difficult.
  • Successful manipulation of the fragment whether derived from blood [Fugimura, Y. et al., J. Biol. Chem.. 261(1), 381-385 (1986), and Fugimura, Y. et al., J. Biol. Chem.. 262, 1734- 1739 (1987)], or expressed from recombinant bacterial host cells.
  • Examples 3 to 6 herein has typically required that a least certain of the cysteine residues thereof be reduced an permanently alkylated or replaced, through mutation, by other amino acids. Without this treatment, undesired reaction of the cysteine residues thereof invariably occurs, leading to the formation of insoluble and biologically inactive polypeptide aggregates unsuited for effective use as therapeutics.
  • residue 449-728 vWF fragments Factors involved in the design of residue 449-728 vWF fragments which avoid these difficulties are as follows. It is known that the residue 449-728 fragment of mature vWF subunit, which contains the platelet glycoprotein Ib( ⁇ ) binding domain, has cysteine residues at positions 459, 462, 464, 471, 474, 509 and 695. It is known also that all of the cysteine residues of the mature vWF subunit are involved in disulfide bonds. (Legaz, et al. , J. Biol. Chem.. 248, 3946- 3955 (1973)). Marti, T. et al. Biochemistry.
  • A a polypeptide comprising reduced and alkylated 52/48 fragment as derived from blood (Fugimura, Y. et al.), or one or more subfragments thereof containing all or part of the GPIb ⁇ -binding domain thereof;
  • polypeptide comprising approximately the residue 441 to 733 fragment of vWF subunit expressed from recombinant bacterial host cells (whether or not containing a disulfide bond linking cysteine residues 509 and 695, see Examples 3-5) , in derivatized or underivatized form, or a subfragment thereof having GPIb ⁇ -binding activity (see, for example, representative species described in
  • (C) a polypeptide comprising approximately the residue 441 to.730 fragment of vWF subunit expressed from recombinant mammalian host cells, as described for example in Example 7, in derivatized or underiv ⁇ atized form, or a subfragment thereof having GPIb ⁇ - binding activity.
  • the above-mentioned polypeptides are the subject of the aforementioned copending '004, '183 and '606 U.S. applications.
  • GPIb ⁇ sequence predicted thereby consists of a 16 amino acid signal peptide, Met" 16 through Pro" 1 , followed by a 610 amino acid mature peptide or polypeptide region. His 1 through Leu 610 .
  • the nucleotide numbering system of Wenger is followed herein.
  • the complete sequence of the aforementioned 45 kDa tryptic fragment of GPIb ⁇ comprises His 1 through Arg 290 or Arg 293 .
  • Representative techniques whereby the His 1 to Leu 610 GPIb ⁇ polypeptide and also the His 1 to Arg 293 fragment thereof (as a His 1 to Ala 302 construct) can be expressed from recombinant mammalian host cells are provided in Example 2.
  • Production of the 45 kDa fragment of GPIb ⁇ from blood has been described previously, Vicente, V. et al., J. Biol. Chem. , 265(1), 274-280 (1990), see also Handa, M. et al. , J. Biol. Chem.. 261(27), 12579-12585 (1986).
  • the structure and properties of GPIb ⁇ are reviewed in Ruggeri, Z.M. , The Platelet Glycoprotein Ib-IX Complex, in
  • Fragments of GPIb ⁇ suitable as domains in the design of the bifunctional molecules or polypeptides of the invention include:
  • polypeptides of (A) above a polypeptide comprising one or more subfragments of a polypeptide of (A) above, and containing all or part of a binding site for thrombins and/or all or part of a binding site for vWF.
  • the above-mentioned polypeptides are the subject of the aforementioned copending '083 and '674 U.S. applications.
  • the invention provides for polypeptides capable of inhibiting the binding of thrombins to GPIb ⁇ receptor on the surface of platelets or-cells, and for bifunctional molecules derived therefrom which are capable of inhibiting the binding of thrombins and/or vWF to said GPIb ⁇ receptor.
  • bifunctional molecules of the invention there is first provided a description of the aforementioned polypeptides capable of inhibiting binding by thrombins.
  • the practice of the invention includes indentification within the amino terminal His 1 to Arg 293 fragment of the GPIb polypeptide of a high affinity binding site for thrombins, and provision of antithrombotic polypeptides patterned thereon that inhibit binding (interaction) of thrombins at this site.
  • Said polypeptides whether in derivatized or underivatized form, comprise subfragments (or combinations subfragments) of the His 1 to Arg 293 fragment of GPIb ⁇ and are believed to function by competing with platelet-bound GPIb ⁇ for thrombins.
  • An additional aspect of the development provides confirmation that the binding of ⁇ -thrombin to platelets can be described by a model based on the existence of three classes of binding sites, those of high affinity (on GPIb ⁇ with a dissociation constant, K d , of about 10" 10 M) , those of medium affinity (K d of about l ⁇ " 8 M) , and of low affinity (K d o about 10 "7 M) (see Harmon, J.T. and Jamieson, G.A. , J. Biol. Chem.. 261, 15928-15933 (1986) for earlier studies) .
  • a further aspect of the development demonstrates that the high affinity binding sites of a platelet for thrombins, located on GPIb ⁇ , perform an important role in a specific platelet activation pathway. Selective inhibition of the high affinity binding site impairs platelet activation. Such impairment can be demonstrated by transient rises in intracellular levels of calcium ions, impairment of dense granule release, and reduction of the amount of fibrinogen binding to platelets and the resultant level of platelet aggregation. However, at sufficient concentration, thrombin can induce platelet activation and aggregation after binding to medium and low affinity receptors.
  • inhibiting the interaction of GPIb ⁇ and thrombins at, for example, thrombin concentrations of less than about InM does not prevent binding by thrombins to the medium and low affinity receptors (it is believed that Vu, T-K. E. et al.. Cell. 64,1057-1068 (1991) have now characterized the independent, medium affinity platelet receptor protein for thrombin) which trigger, independently, activation of platelets at higher thrombin concentrations.
  • the polypeptides of the invention are effective as antithrombotics in that they inhibit binding of thrombins to GPIb ⁇ , said binding being important to platelet activation in the early stages of a hemostatic response to a wound or to a diseased vessel. Accordingly, one of the effects of the polypeptides of the invention may be to inhibit the docking of thrombins at the high affinity sites on the platelet surface so that thrombins cannot be oriented to interact with additional components of the hemostatic mechanism.
  • Such regulation is not expected to interfere with participation of thrombin in the clotting cascade when fully activated (such as by a major wound) or at sites in the body removed from where a -therapeutic composition is targeted (such as to a site of surgery) .
  • Synthetic polypeptides in underivatized form, patterned upon all or part of the residue 237 to 291 sequence of GPIb ⁇ were tested for activity as inhibitors of the interaction between platelet GPIb ⁇ and thrombins.
  • This region of GPIb ⁇ was selected for testing because it defines the epitope of LJ-IblO monoclonal antibody (described below) which is known to inhibit thrombin-GPIb ⁇ interaction.
  • Preparation of synthetic peptides and methods to test their ability to inhibit binding of thrombins to GPIb ⁇ are provided in Example 1. As shown in Figure 1, synthetic peptides containing (or overlapping with) residue sequence Leu 275 to Glu 285 had inhibitory activity.
  • Representative operable polypeptides include Gly 271 to Glu 285 (SEQ ID NO: 3), Pro 265 to Glu 285 (SEQ ID NO: 2), and Asp 269 to Glu 282 (SEQ ID NO: 10).
  • Pro 265 to Tyr 279 (SEQ ID NO: 5) is ineffective as an inhibitor polypeptide. Comparative results are shown in Figure 2 (see also Example 1) for the polypeptide Val 236 to Lys 253 and the aforementioned Pro 265 to Glu 285 polypeptide.
  • the thrombin-binding region of GPIb ⁇ is within residues 265 to 285 thereof and in particular, within residues 275 to 285, and/or polypeptides overlapping therewith, wherein are contained the 3 tyrosine residues 276, 278 and 279 ( Figure 1) . It has been determined that one or more of these tyrosines are sulfated on the phenolic oxygen of the tyrosine ring. Posttranslational modification of tyrosine by sulfation has been observed also at the binding sites for thrombin of other thrombin-interactive proteins. Accordingly, the invention includes also within its scope sulfated forms (at tyrosine) of aforementioned GPIb ⁇ polypeptides.
  • the 45 kDa amino terminal fragment of GPIb ⁇ containing a Leu 275 to Glu 285 sequence therein, is also effective as an inhibitor of thrombin-GPIb ⁇ interaction.
  • Bifunctional Molecules of the Invention is the recognition that the binding domain of GPIb ⁇ for vWF and of GPIb ⁇ for thrombins are in close proximity on the GPIb ⁇ polypeptide. This high affinity binding site for thrombins is defined, at least in part, by the Leu 275 to Glu 285 fragment of GPIb ⁇ and wherein, as expressed in human cells, one or more of Tyr 276 , Tyr 278 and Tyr 279 are likely sulfated.
  • the binding site for vWF on GPIb ⁇ is located on the amino terminal His 1 to Arg 293 (45 kDa) fragment of GPIb ⁇ .
  • vWF and thrombin binding sites are distinct as evidenced by the behavior of LJ-Ibl and LJ-IblO monoclonal antibodies (Handa, M. et al., J. Biol. Chem. , 261, 12579- 12585 (1986) .
  • LJ-Ibl completely inhibits vWF-GPIb ⁇ interaction but has no or minimal effect on the binding of thrombins to platelets whereas LJ-IblO, which interferes with GPIb ⁇ -thro bin interaction, has no effect on vWF-GPIb ⁇ interaction.
  • antithrombotic bifunctional molecules can be constructed comprising two linked domains, a first domain capable of inhibiting the binding of thrombins to platelet glycoprotein Ib ⁇ and a second domain capable of inhibiting the binding of vWF to glycoprotein Ib ⁇ .
  • the importance of the present development is based upon the recognition that platelets can become activated (and then aggregated) by more than one pathway or type of stimulus. Providing a therapeutic composition which has the capability of inhibiting more than one such pathway is of substantial clinical importance.
  • the bifunctional molecules of the invention provide such a therapeutic advantage by inhibiting thrombin-induced and vWF-induced platelet activation, two of the most important of the activation pathways.
  • Substantial therapeutic advantage can be attained if both of the above- mentioned activation pathways (through thrombins and vWF) were to be inhibited simultaneously. Such advantage is expected to be greater than that provided by the use of separate or dissociable therapeutic molecules (not covalently bonded to each other) .
  • vWF binding to GPIb ⁇ is predicated on the identification of domains that interact with (bind to) the GPIb ⁇ polypeptide and prevent vWF from gaining access to or remaining on the receptor site thereof.
  • Representative domains are the following. Polypeptides, whether derivatized or underivatized, that comprise approximately the Arg 441 to Val 733 fragment of the vWF subunit or one or more subfragments thereof containing all or part of the GPIb ⁇ -binding site thereof.
  • Identification of particular subfragments of vWF subunit that contain all or part of a binding site of vWF for GPIb ⁇ , and that are suitable as domains in this embodiment of the invention, may be determined by one or more assays well known in the art, including the capability of a domain to inhibit the binding of LJ-Ibl monoclonal antibody to platelet GPIb ⁇ , or to inhibit ristocetin or botrocetin-induced binding of I25 I-labelled multimeric vWF to formalin-fixed platelets.
  • preferred species of subfragment of vWF subunit are provided in Example 6. Additional domains effective in the practice of the invention include mutant vWF polypeptide sequence having enhanced affinity, relative to wild type for GPIb ⁇ , and "derivatives" of vWF polypeptide sequence, as that term has been specially defined herein.
  • a domain is an antibody, or an active fragment thereof, having as its epitope, or as part thereof, all or part of the vWF binding site of glycoprotein Ib ⁇ .
  • Use of a complete antibody molecule is consistent with the practice of the invention as long as the patient is monitored for adverse clinical effects resulting from an immune response thereto including those effects mediated by the F c region of the antibody.
  • Preferred species see Example 6 for methods of constructing same) .
  • Example 8 describes how active fragments of the LJ-Ibl0 antibody (reactive to the binding site of GPIb ⁇ for thrombins) suitable as domains in the practice of the invention may be prepared.
  • Factors that are important in the design of such active fragments for use in the practice of the invention include
  • F v fragments (Example 8) or single polypeptide or peptide fragments thereof containing all or part of one or more CDR regions thereof provide effective inhibition peptides.
  • the domain of the bifunctional molecules capable of inhibiting binding of thrombins thereto it is noted that such a domain may function in one of two ways: Antibodies, or active fragments thereof that bind to the thrombin binding site of GPIb ⁇ prevent thrombins from from occupying the GPIb ⁇ site and activating the underlying platelet.
  • Factors useful in the selection of an antibody or an active fragment thereof directed to the vWF-binding site of GPIb ⁇ are equally applicable to the selection of antibodies or active fragments thereof directed to the thrombin-binding site (such as of LJ-IblO) .
  • An additional type of molecule suitable as a domain to inhibit binding of thrombins to GPIb ⁇ is represented by the amino terminal His 1 to Arg 293 fragment of GPIb ⁇ or a fragment thereof containing all or part of the thrombin binding site thereof. Without limitation as to theory, it is believed that anti-thrombin domains of this type direct thrombins to the domain of the bifunctional molecule patterned on GPIb ⁇ instead of the underlying GPIb ⁇ polypeptide receptor of the platelet, thereby avoiding platelet activation.
  • Mutant and derivatized peptides as those terms have been previously described are also useful in the design of the anti-thrombin domain. Screening of mutant GPIb ⁇ -derived or antibody-derived polypeptides for enhanced activity to inhibit binding of thrombins to GPIb ⁇ may be accomplished using the below described mutagenesis strategy and the assay procedure of Example 1. It is noted that the order of the domains with respect to each other is not important in the design of the bifunctional molecule as long as the linkage thereof is of such design and length so as to provide sufficient flexibility for both domains of the molecule to function.
  • the molecules in the above embodiments of the invention are described as "bifunctional," multifunctional or polyfunctional molecules having three or more binding functions are within the practice of the invention and have utility therein. The domains utilized in the practice of the invention have also such additional functions.
  • the domains of the bifunctional molecules must be linked.
  • the linkage is covalent and may be accomplished according to either of two basic procedures, each of which is readily accomplished and well known in the art.
  • Encoding DNA for one domain can be combined with encoding DNA for the other domain in a continuous single DNA construct using well known recombinant techniques leading to the expression of a single polypeptide having two domains (see Example 8 for representative methodology) . If this method is adopted, it is highly preferred that the encoding DNA encode also a linker peptide sequence between the two polypeptide domains. Such a sequence facilitates each domain adopting a nativ?. tertiary conformation,, or having sufficient freedom to find and bind to its target site ithout steric interference from the other domain. Linkers comprising between approximately 10 and 50 amino acids are most preferred but can be optimized for each individual case with appropriate experimentation.
  • the two domains can be expressed or otherwise provided separately and then chemically linked by attaching between them a spacer or linker, again preferrably of suitable length to avoid steric interference between the domains.
  • the two domains can be covalently linked with a linker using attachment at suitable representative reactive groups. Such reactive groups may preexist in the original structure of the polypeptide domains or be covalently added after the isolation or production of the polypeptides.
  • a representative linker suitable for use either according to method (A) or method (B) comprises a series of glycine residues allowing a high degree of conformational flexibility between the two domains.
  • the necessary length of the linker may be determined by appropriate experimentation.
  • each including both domains of the bifunctional molecule, mutants of the amino acid sequences which confer thereon enhanced binding affinity for the target receptor(s) .
  • mutants of the amino acid sequences which confer thereon enhanced binding affinity for the target receptor(s) .
  • a method for randomly producing mutant vWF-encoding DNA sequences and for screening the resultant expressed polypeptides for those which have enhanced binding affinity for GPIb ⁇ Similar approaches, based on cloning methodology well known in the art, can be designed to express and then screen mutants of GPIb ⁇ polypeptide for enhanced affinity as domains in the practice of the invention.
  • novel variant DNA sequences can be constructed which encode variant vWF-derived polypeptides.
  • the resultant potential therapeutic polypeptides can be screened for relative binding affinity: (1) in direct binding assays for affinity to GPIb ⁇ ; (2) in botrocetin- or ristocetin-induced binding assays; or (3) to conformation dependent vWF-specific antibodies. Random mutagenesis experiments can also be performed using vWF DNA constructs suitable for expression in mammalian cells such as those of Example 7.
  • Mutant oligonucleotides suitable for site directed mutagenesis protocols and spanning sequential 10 amino acid subdomains of the loop can be generated using a procedure designed to yield a randomly mutagenized oligonucleotide population.
  • the randomized vWF oligonucleotide is then hybridized, for example, to M13mpl8 to copy the mutation into a residue 441-733 encoding DNA sequence.
  • a random oligonucleotide population suitable for causing permutation of the residues between positions 690 and 710 of the mature vWF subunit can be constructed as follows.
  • the oligonucleotide corresponds to transcribed strand DNA.
  • (A,T,G,C) for oligonucleotide synthesis is "doped" with a small amount of each of the other three bases. Incorporation of one of the "doping" nucleotides results in a mutant oligonucleotide. The amount of doping can be adjusted to control results.
  • the resultant randomized population of mutant oligonucleotides is then used in the standard site directed mutagenesis protocol (Example 3) to construct a pool of mutagenized vWF "loop" DNA sequences in M13mpl8 corresponding to the mature vWF subunit residue 441-733 fragment and suitable for subcloning into a bacterial expression system.
  • the resultant mutant M13mpl8 populations are then subject to restriction, and the mutagenized DNA sequences are inserted into vectors or plasmids such as pET-3A for expression in host bacterial cells.
  • vectors or plasmids such as pET-3A for expression in host bacterial cells.
  • Large scale screening of mammalian clones is generally much more difficult than for bacterial clones.
  • promising mutations identified in bacterial constructs may later be inserted into mammalian or other eucaryotic host cells for further testing or for commercial-scale polypeptide production.
  • the mutant clones can then be screened in GPIb ⁇ binding assays or in binding assays with vWF-specific monoclonal antibodies.
  • Mutant clones having cell lysates which exhibit enhanced platelet binding or antibody response can be sequenced to determine the amino acid alteration(s) responsible for the mutant phenotype. In this way, a very systematic analysis of the loop region of vWF can be performed and mutations, which alter the binding of vWF to GPIb ⁇ can be identified.
  • the mutagenesis technique is particularly applicable to permuting the amino acid sequence regions of the mature subunit believed to contain actual GPIb ⁇ binding (interaction) sites for the purpose of enhancing their GPIb ⁇ affinity. Screening of Mutant vWF-Derived
  • a device used for the enzyme- linked immunofiltration assay technique can be adapted in combination with immobilization of the mutant vWF-derived polypeptides to be tested. It is considered most efficient to test initially the effect of mutant codons on vWF polypeptides expressed from bacterial constructs and to then copy potentially useful mutations (using, for example, mutagenesis in M13mpl8 vehicle) into a mammalian expression construct. High levels of mutant vWF polypeptides corresponding to mutant DNA sequences can be expressed from pET-3A type bacterial expression plasmids such as p5E. Mutant polypeptides constitute a major portion of host E.coli cell lysates and can be screened readily for GPIb ⁇ affinity.
  • ELIFA enzyme- linked immunofiltration assay technique
  • site directed mutagenesis can be performed following the procedure of Example 3, using as template in M13mpl8 the vWF fragment corresponding to p5E expression plasmid which because of the use of BamHl linkers in assembly of p5E is recovered therefrom and inserted into M13mpl8 as an Xbal/Hindlll fragment.
  • oligonucleotide pool oligonucleotides each having randomly mutagenized residue 690 to 710 sequences are used.
  • the mutagenized population of M13mpl8 constructs can be cloned into pET-3A plasmids after which the expression plasmids can be transformed into E.coli BL21 (DE3) .
  • Preparation of mutant polypeptide extracts from E.coli BL21(DE3) for screening follows the procedure of Example 3, with the final step being solubilization of extracted inclusion body material with 8 M urea at room temperature for 2 hours.
  • Resultant extracts of expressed mutant p5E-type vWF polypeptides are immobilized following the manufacturer's instructions onto a nitrocellulose membrane (0.45 ⁇ pore size) using 96-well sample application plates (Easy-Titer ® ELIFA System, Pierce Co., Rockford, IL) and a vacuum chamber.
  • 96-well sample application plates Easy-Titer ® ELIFA System, Pierce Co., Rockford, IL
  • Commercially available pump materials can be used.
  • the apparatus is suitable for screening large series of clone lysates in an ELIFA or dot blot system and allows also quantitative transfer of sample fluids to underlying microtiter wells without cross contamination.
  • Immobilization of the vWF polypeptides is accomplished by causing a suitable volume, such as 200 ⁇ l, of each resuspended inclusion body pellet material (in 8 M urea) to be vacuum-drawn through the individual wells to the nitrocellulose membrane over a 5-minute period. Several 200 ⁇ l volumes of Hepes-buffered saline are then drawn through the membrane to remove urea. The protein binding capacity of the membrane is then saturated by passing through it three consecutive 200 ⁇ l aliquots of HEPES/BSA buffer having a pH of 7.4 and comprising 20 mM Hepes, 150 mM NaCl, and 1% w/v bovine serum albumin (Calbiochem, La Jolla, CA) .
  • a suitable volume such as 200 ⁇ l
  • a 50 ⁇ l volume of HEPES/BSA containing botrocetin (at approximately 0.5 ⁇ g/ml) or containing ristocetin ( at approximately 1 mg/ml) can be vacuum drawn through the nitrocellulose membrane again over a 5-minute period.
  • the ristocetin-induced precipitation of bacterially-expressed vWF polypeptides observed under some test conditions is not expected to cause difficulty in this assay as the polypeptide is already immobilized.
  • GPIb( ⁇ ) represented by its external domain, glycocalicin, or the 45 kDa tryptic fragment thereof is next applied to the nitrocellulose using the vacuum system and the 96-well-plate.
  • the GPIb ⁇ •fragments are purified and I25 iodinated by standard procedures (Vicente, V. et al., J. Biol. Chem.. 265, 274-280 (1990)). Fifty ⁇ l aliquots of HEPES/BSA containing 125 I-GPIb ⁇ fragments (0.25 ⁇ g/ml having a recommended specific activity of between approximately 5xl0 8 and approximately 5xl0 9 cpm/mg) can then be vacuum drawn through the nitrocellulose filter over 5 minutes. The membrane is then allowed to dry and discs corresponding to the position of each application well are cut out and counted in a scintillation spectrometer to determine bound radioactivity.
  • An autoradiograph of the membrane can also be obtained before cutting out the discs in order to ascertain that there was no leakage of radioactivity from one well to another.
  • the counting process may be facilitated by scanning the developed autoradiogram in a densitometer to digitize the intensity of developed spots. As long as the autoradiogram is not excessively overdeveloped, beyond the linear region of response, useful qualitative results are obtained.
  • the cells are harvested by centrifugation for 1 minute at 10,000 g and then washed and repelleted (at 10,000 g) 3 times with phosphate buffered saline (0.14 M NaCl, 0.1 M Na 2 HP0 4 pH 7.0).
  • the bacterial pellet is then solubilized by boiling for 10 minutes in a buffer comprising 0.01 M NaH 2 P0 4 , 10 mM Na 2 EDTA, 1% (w/v) sodium dodecylsulfate, pH 7.0.
  • the incubation is continued for 2 hours at 60°C in the presence also of 10 mM dithiothreitol (DTT) .
  • DTT dithiothreitol
  • Suitable volumes (such as 200 ⁇ l) of such extracts can be used directly in ELIFA apparatus or dot immunoblot analyses.
  • vWF derived polypeptides from colonies representing the most intense response are selected for confirmation of enhanced binding using methods such as subjecting purified or partially purified extracts therefrom as appropriate to: (A) immunoblotting with an appropriate antibody; (B) assaying for ability to inhibit botrocetin-induced vWF binding to formalin-fixed platelets on a dose dependent basis; or (C) assayed for ability to inhibit the binding of anti GPIb ⁇ monoclonal antibodies to platelets.
  • Clones which confer enhanced positive responses in these systems are then subjected to standard DNA sequencing procedures to identify the vWF gene mutations responsible for the mutant properties.
  • the appropriate mutations may be copied into a vWF DNA sequence within a plasmid (such as a pADS/WT-pCDM ⁇ 1100 expression plasmid) suitable for expression in CH0-K1 cells (see Azuma, H. et al., J. Biol. Chem. ,
  • cysteine residues thereof have been inactivated (my missense mutation with site-directed mutagenesis) so as to avoid undesired crosslinking
  • cysteine residues thereof have been inactivated (my missense mutation with site-directed mutagenesis) so as to avoid undesired crosslinking
  • cysteine residues There are available also numerous other well known procedures that can be used to inactivate cysteine residues.
  • One such technique involves treatment of cysteine residues with a reducing agent such as, for example, 6-mercaptoethanol or dithiothreitol "DTT" followed by permanent alkylation (for example, with iodoacetamide) .
  • covalent labels may be attached to the target cysteine residues, the only requirements being that the label be applied under pH conditions which do not irreversibly denature the target protein.
  • the attachment is of a kind which, under the conditions to which the fragment is exposed during further processing or storage, will not allow chemical reaction with other cysteine residues.
  • covalent labelling procedures are generally known in the art and include also, for example, reaction with (A) iodoacetic acid or (B) iodinating agents such as iodofluorescein. Alteration can be accomplished also by site directed mutagenesis of an encoding DNA or by deleting sequence positions corresponding to cysteine.
  • bifunctional molecules and of the polypeptides of the invention has been described in terms of achieving, as an example, a beneficial antithrombotic response in the presence of low concentrations of thrombin without preventing platelet response to higher concentrations of thrombin. Accordingly, administration of a GPIb ⁇ receptor-selective composition to a patient (for example, undergoing surgery or afflicted with thrombosis) would not only provide desired localized or immediate antithrombotic effects, but could minimize the risk of systemic antihemostatic effects that would prevent response to higher concentrations of thrombin associated with a major bleeding event that would fully activate the clotting cascade, or response at sites in the body far removed from the initial target site.
  • GPIb ⁇ is not limited, in approach, solely to inhibition of the binding of thrombins or vWF at GPIb ⁇ sites on platelets.
  • Expression of GPIb ⁇ is not limited to platelets or to the megakaryocytic cells from which platelets are produced. It has been determined that GPIb ⁇ is also expressed in endothelial cells - Konkle, B.A. et al., J.
  • GPIb ⁇ or molecules with homologous structure and function, are also present on other normal and pathological (including cancerous) cells.
  • Asch, A.S. et al. , J. Clin. Invest.. 81, 1600-1607 (1988) have reported identification of a GPIb-like protein in human smooth muscle cells.
  • Grossi, I.M. et al. , FASEB J.. 2, 2385-2395 (1988) have reported that certain carcinoma cell lines express proteins which react with anti-GPIb monoclonal antibodies. These antibodies were also reported to inhibit tumor- associated platelet aggregation.
  • the bifunctional molecules or polypeptides of the invention have utility in modulating the response to thrombins and/or vWF of.cells in blood vessel walls, such as endothelial cells or smooth muscle cells. This is expected to have clinically useful consequences, both for the short term and long term response of vessels to thrombogenic and atherogenic stimuli.
  • endothelial cells stimulated by cytokynes at a site of inflammation or as a consequence of an immune-mediated insult may express GPIb ⁇ (or molecules with homologous structure and function) .
  • the binding of vWF and/or thrombins to the expressed receptor ma lead to further activation of the endothelial cells and/or mediate the attachment of platelets to these cells.
  • Platelets become activated as a consequence of adhesion and, thus release growth factors and other factors that may initiate the proliferation of smooth muscle cells in the vessel wall.
  • Smooth muscle cells may also be further activated as a consequence of thrombin binding to expressed GPIb ⁇ , or molecules with homologous structure and function.
  • thrombin bound to endothelial cells represents a potent catalyst for a series of biochemical enzymatic reactions important for the formation of fibrin, fibrin deposition on the vessel wall may have a pathogenetic role both in the long term development of atherosclerotic lesions as well as in acute occlusive events. The latter may take place in vessels stenosed by advanced atherosclerotic processes, usually as a consequence of rupture of an atherosclerotic plaque but, possibly, also following adhesion of platelets to damaged and stimulated endothelial cells.
  • thrombin and/or vWF binding to endothelial cells and other cells of the vessel wall may influence the onset, progress and terminal occlusive events of atherosclerotic diseases.
  • inhibition of thrombin binding to GPIb ⁇ or homologous molecules may have an indirect beneficial antithrombotic effect by favoring the binding of thrmbins to thrombomodulin. After the latter interaction takes place, thrombin itself acquires an antithrombotic activity through the activation of the Protein C pathway of inhibition of coagulation cofactors.
  • inhibition of the binding of thrombins or vWF to GPIb ⁇ receptors on tumor cells can inhibit certain behaviors of tumor cells, thereby influencing their metastatic potential.
  • platelets interact with tumor cells and influence their ability to penetrate the vessel wall and colonize tissues, the central event in the establishment of a metastatic are of tumor growth.
  • the assembly of fibrin around tumor metastasis may be one of the mechanisms by which tumo cells protect themselves from defense mechanisms of the hos organism after seeding new tissues. Consequently, the inhibition of the binding of thrombins and vWF to GPIb ⁇ or homologous molecules on tumor cells may have clinical advantages in the treatment of cancer patients by limiting the ability of primary tumors to grow and metastatize.
  • Additional Antibodies with Therapeutic Activity Antibodies, and particularly conformation dependent antibodies are powerful tools for analyzing the structure and function of macromolecules. By blocking macromolecular interactions, antibodies can also have important therapeuti and diagnostic utility.
  • this invention includes within its scope antibodies that are specific for all or part of any of the bifunctional molecules or polypeptides of the invention, including, therefore, antibodies that recognize idiotypes, all said antibodies being made by a process which involves immunizing animals with a bifunctional molecule, or polypeptide, of the invention or a component thereof.
  • Therapeutic compositions One or more of the bifunctional molecules or polypeptides of the present invention can be formulated into pharmaceutical preparations for therapeutic, diagnostic, or other uses.
  • compositions are dissolved in water typically containing also physiologically compatible substances such as sodium chloride, glycine, and the like resulting in a solution -having a pH, ionic strength, and osmotic potential compatibl with therapeutic use (such concentrations being well known i the art) , said water and any other physiologically compatibl substances comprising a pharmaceutically acceptable carrier.
  • physiologically compatible substances such as sodium chloride, glycine, and the like
  • the amount to administer for the prevention or inhibition of thrombosis will depend upon the affinity of the molecule for platelet GPIb ⁇ in vivo, and/or for other macrocmolecules that participate in hemostasis and thrombosis in the body, and on the severity with which the patient is subject to thrombosis Said amount can be determined readily for any particular patient.
  • a therapeutic composition containing one or more of the bifunctional molecules or polypeptides of the invention and also additional therapeutic substances.
  • additional substances include heparin and other anticoagulants, aspirin or other antiplatelet drugs, or tissue plasminogen activator or other prefibrinolytic drugs.
  • Example 1 Inhibition of ⁇ -thrombin binding to platelets by synthetic peptides
  • This example demonstrates the effect of two synthetic peptides (patterned upon the amino acid sequence of GPIb ⁇ ) o the binding of ⁇ -thrombin to platelets.
  • the peptides were synthesized chemically following the method of Houghten,
  • Washed platelets were suspended at a concentration of 1.875 10 12 /liter in binding buffer, hereinafter (136 mM NaAcetate, 25 mM Tris base, 0.6% (w/v) polyethyleneglycol 6,000, pH 7.3) and incubated at 37°C for 10 minutes immediately prior to use. There were also prepared the following stock solutions:
  • BSA bovine serum albumin
  • the assay is then performed by adding, in rapid succession, 50 ⁇ l binding buffer; 20 ⁇ l of 4.1% BSA in binding buffer; 20 ⁇ l washed platelet suspension (final assay count, 3 x 10 n /liter) ; 25 ⁇ l of peptide stock (after dilution resulting in peptide assay concentrations of 50, 100, 200, 400, etc. ⁇ M) in "Hepes buffer” or 25 ⁇ l Hepes buffer as the control; and finally 10 ⁇ l 125 I- ⁇ -thrombin stock solution (D) resulting in a final ⁇ -thrombin concentration of 0.46 nM.
  • platelet-bound and free ⁇ - thrombin were separated by centrifugation of the platelets (for 4 minutes at 12,000 g through a 20% sucrose gradient in Hepes buffer consisting of 20 mM Hepes, 150 mM NaCl, pH 7.3 " ).
  • the radioactivity associated with the platelet pellet was counted in a y scintillation spectrometer, and bound ⁇ - thrombin in the mixtures containing peptide was expressed as percentage of that bound to platelets in the control mixture (without peptide, as shown on the ordinate of Figure 2) .
  • the average inhibitory effect of Pro 265 -Glu 285 corresponds to approximately 64% inhibition of the total amount of ⁇ - thrombin otherwise bound to the platelets, said incomplete inhibition being consistent with the fact that platelets possess more than one class of thrombin binding sites.
  • a concentration of 80 ⁇ M of the peptide was needed to achieve approximately 50% of inhibition under the assay conditions used.
  • the Val ⁇ -Lys 253 peptide had no effect as a binding inhibitor under these assay conditions ( Figure 2 shows one representative trial for this peptide; the average of four trials being 149% of control) .
  • Peptide Phe 216 -Thr 240 achieved a negligible inhibitory effect, approximately 10%, at the 400 ⁇ M concentration.
  • Step 1 Construction of a DNA sequence for expression of the mature His ⁇ Leu 61 " polypeptide
  • flanking oligonucleotides were synthesized for the amplification in a polymerase chain reaction of a region of the GPIb ⁇ gene which it was believed would be suitable as a probe to screen a human genomic lambda ( ⁇ ) phage library. Accordingly, human genomic DNA was subjected to enzymatic amplification in a polymerase chain reaction according to the method of Saiki et al., Science. 239, 487- 491 (1988) . The procedure utilizes a double stranded GPIb ⁇ DNA sequence, a subsegment of which is to be amplified, and two single stranded oligonucleotide primers which flank the ends of the subsegment.
  • the primer oligonucleotides (in the presence of a DNA polymerase and deoxyribonucleotide triphosphates) were added in much higher concentrations than the DNA to be amplified.
  • PCR reactions were performed with a DNA thermal cycler (Perkin Elmer Co., Norwalk, CT/Cetus Corporation, Berkeley, CA) using Taq polymerase (Thermus aguaticus) .
  • the reactions were run in 100 ⁇ t volumes containing 1.0 ⁇ g of human genomic DNA, 1.0 ⁇ g of each synthetic oligonucleotide primer, and buffer consisting of 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 0.1% gelatin (BioRad Co., Richmond, CA) and 200 mM of each dNTP.
  • PCR conditions were 35 cycles of 30 seconds at 94°C, 30 seconds at 52°C and 1 minute at 72°C.
  • Amplified fragments were then purified and isolated by electrophoresis through a 2% agarose gel, Maniatis et al.. Molecular Cloning, A Laboratory Manual. 164-170, Cold Spring Harbor Lab., Cold Spring Harbor, NY (1982) .
  • oligonucleotides were synthesized by the phosphoramidite method, Sinha et al. , Tetrahedron Letters. 24, 5843 (1983) using a model 380B automated system, Applied Biosystems, Foster City, CA.
  • oligonucleotides selected were:
  • Oligonucleotide (A) (SEQ ID NO: 11)
  • Oligonucleotide (B) (SEQ ID NO: 12) 3' - GC GAC GGA GAA CCA CGG GAC - 5' t r
  • Oligonucleotide (A) is equivalent to non-transcribed strand DNA (coding strand) for nucleotides 644-674 (using the numbering system of Wenger et al.-, Biochem. Biophys. Res.
  • Oligonucleotide (B) is shown 3 ' ⁇ 5' and is equivalent to the transcribed strand (noncoding DNA) .
  • the corresponding coding strand is shown 5' ⁇ 3' in lower case letters. Nucleotide positions are according to Wenger et al.
  • T 4 kinase was used to add phosphate groups to each end of the amplified fragment.
  • T 4 ligase was used to blunt end ligate the fragment into the Smal site within the multiple cloning sequence of the double stranded replicative form of M13mpl8 bacteriophage.
  • the ability to isolate a stable single stranded (+) form of the virus is particularly useful to verify the integrity of any cloned sequences therein. See Messing, J. Meth. Enzvmologv. 101, 20-78 (1983), and Yanish- Perron et al., Gene. 33, 103-109 (1985).
  • the GPIb ⁇ DNA insert was completely sequenced using single stranded dideoxy methodology (Sanger et al., Proc. Natl. Acad. Sci. USA. 74, 5463-5467 (1977) utilizing the single stranded (+) form of M13mpl8. Sequencing in M13mpl8 established that the GPIb ⁇ insert was 301 base pairs in length, indicating that the corresponding cDNA region, Lopez et al., did not involve an intron boundary. The 301 base pair (bp) fragment was then subjected to nick translation for incorporation of 32 P-labelled nucleotides, thus converting the fragment into a radiolabelled probe, Rigby et al., J. Mol. Biol.. 113, 237 (1977).
  • a human genomic ⁇ phage library (using Lambda FixTM, Stratagene, La Jolla, CA) was prepared using an EcoRI partial digest of human cell DNA. The library was screened following the hybridization and plaque purification procedure of Benton et al.. Science. 196, 180-182 (1977) using E. coli strain LE 392 as host. Screening with the 301 bp fragment resulted in the isolation of 6 positive clones after 4 cycles of plaque purification.
  • the approximate 6000 base pair fragment visualized and extracted from an agarose gel was then cloned into pBluescript KS- plasmid (Stratagene Co., La Jolla, CA) at its EcoRI site.
  • the plasmid was then propagated in E. coli strain XL-1 Blue (Stratagene Co.). Plasmids were recovered from host E. coli by an alkaline cell lysis procedure, Birnboim and Doly, Nucleic Acids Research, 7, 1513 (1979) followed by purification by CsCl/ethidium bromide equilibrium certtrifugati ' on according to Maniatis et al., at 1.42.
  • Plasmid so isolated was then digested with BamHl and Bglll creating a 2161 base pair fragment (nucleotides 503 to 2663 using the numbering system of Wenger et al.) which fragment extends from upstream above the initiating MET 1 codon (nucleotides 537-539) to downstream below the ' LEU 610 codon (nucleotides 2412-2414) and the TGA translation stop codon (2415-2417) .
  • the BamHl site of the fragment corresponds to nucleotides 502-507 and the Bglll site thereo nucleotides 2658-2664.
  • the 2161 bp fragment was then cloned into the BamHl sit of pBluescript KS- (Strategene Co., La Jolla, CA) as a BamHI Bglll fragment. Since BamHl and Bglll restriction sites contain identical internal sequences GATC/CTAG, a Bglll restricted site may be annealed into a BamHl site. The fragments were ligated with T 4 DNA ligase, however the integrity of the affected Bglll end was not restored. Hybridization with the 301 base pair probe and sizing on agarose were repeated. The plasmids were propagated in E. coli XL-1 Blue.
  • pCDM8 vector developed by Seed et al., Nature. 329, 840-842 (1987) and available from Invitrogen, San Diego, CA
  • Dr. Timothy O'Toole Scripps Clinic and Research Foundation, La Jolla, CA to include a neomycin resistance gene (phosphotransferase II) that was cloned into the BamHl restriction site of pCDM ⁇ as a part of a 2000 base pair BamHl fragment.
  • the protein produced by the neomycin (neo) gene also confers resistance against other aminoglycoside antibiotics such as Geneticin ® G418 sulfate (Gibco/Life Technologies, Inc., Gaithersburg, MD) .
  • neomycin resistance markers are commercially available. Examples include pcDNA 1 ⁇ ⁇ (Invitrogen, San Diego, CA) , Rc/CMV (Invitrogen, San Diego, CA) and pMAM nco (Clontech, Palo Alto, CA) . If necessary the GPIb ⁇ fragment may be differently restricted or modified for expression capability in these other expression plasmids.
  • XhoI-NotI fragment from pBluescript KS- plasmid was inserted into pCDM8 neo which had been restricted with Xhol and Notl.
  • Ampicillin sensitive E. coli strain XS-127 cells (Invitrogen, La Jolla, CA) were transformed with the resultant ligated DNA mixture following the method of Hanahan, J. Mol. Biol.. 166, 557-580 (1983). Plasmids from resultant colonies were characterized by restriction mapping and DNA sequencing to identify colonies which contained the intended insert.
  • One such plasmid (designated pMWl) , was maintained in E. coli strain XS-127, and was selected for mammalian cell transformation procedures.
  • supercoiled plasmids Prior to use in transforming mammalian cells, supercoiled plasmids (pMWl) were recovered from host E. coli by the alkaline cell lysis procedure of Birnboim and Doly followed by purification by CsCl/ethidium bromide equilibrium centrifugation according to Maniatis et al., at 1.42.
  • Step 3 Transformation of Chinese hamster ovary cells pMWl was introduced into CHO-K1 Chinese hamster ovary cells (ATCC-CCL- 61) by a standard calcium phosphate-mediated transfection procedure. Chen et al., Mol. Cell. Biol.. 7(8), 2745-2752 (1987) .
  • CHO-K1 cells were grown to confluence at 37°C in Dulbecco's modified Eagle's medium (DMEM) (Gibco/Life Technologies, Inc. , Gaithersburg, MD) supplemented with 10% heat-inactivated fetal calf serum (FCS, Gibco) , 0.5 mM of each nonessential amino acid (from NEAA supplement,
  • DMEM Dulbecco's modified Eagle's medium
  • FCS heat-inactivated fetal calf serum
  • CHO-K1 cells have a doubling time in DMEM/10% FCS of approximately 16 hours under- these conditions.
  • pMWl plasmids were recovered from cultures of E. coli strain XS-127, according to the method of Birnboim and Doly, Nucleic Acids Research. 7, 1513 (1979) as described above. Ten ⁇ g of plasmids were applied to the cells of each 60 mm dish in a calcium phosphate solution according to the method of Chen et al. After inoculation with plasmid the cells were maintained in DMEM/10% FCS at 37°C in a 5% C0 2 atmosphere.
  • the cells were trypsinized as follows. Growth medium for each dish was replaced by 3 mf of a solution of phosphate-buffered saline (37 mM NaCl, 27 mM KC1, 4.3 mM Na 2 HP0 4 - 7H 2 0/1.4 mM K 2 HP0 4 , pH 7.4) containing also 0.25% trypsin, 0.2% (w/v) EDTA. Trypsinization was conducted for 3 minutes. The trypsin- containing medium was removed and the dishes were then place in the incubator for a further 15 minutes after which the cells were resuspended in DMEM containing 10% FCS.
  • phosphate-buffered saline 37 mM NaCl, 27 mM KC1, 4.3 mM Na 2 HP0 4 - 7H 2 0/1.4 mM K 2 HP0 4 , pH 7.4
  • trypsinization was conducted for 3 minutes. The trypsin- containing medium was removed and the dishes
  • the cell from each dish were then split 20 fold, and plated at a density of approximately 1.2 x 10 4 cells/60 mm dish (approximately 2% of confluence) .
  • Production of stable transformants, which have integrated the plasmid DNA was then accomplished by adding Geneticin ® G418 sulfate to the 60 mm dishes to a concentration of 0.8 mg/mf. Growth was continued for 14 day at 37°C in a 5% C0 2 atmosphere. Surviving independent colonies were transferred to 12 well plates using cloning rings and then grown for another seven days in DMEM/10% FCS supplemented with 0.8 mg/mf of Geneticin ® . Under these conditions 3 to 7 surviving colonies per plate were apparent after 10-14 days. Approximately 100 stable transformants ca be isolated from each original 60 mM dish originally containing approximately 5 x 10 5 cells at a plate density of approximately 70% of confluence.
  • Colonies expressing the recombinant mature GPIb ⁇ polypeptide were detected by dot-blot analysis on nitro ⁇ cellulose after lysis in buffer. As a control, recombinant cell extracts were compared with that from nontransfected CHO-K1 cells.
  • non-transfected or transfecte CH0-K1 cells were harvested with 3.5 mM EDTA and resuspended in 0.25 M Tris-HCl pH 7.5 (10 3 cells/ ⁇ £) .
  • Cells were lysed by three cycles of freezing and thawing and centrifuged at 12,000 g to remove cell debris. The resulting supernatant was kept at -70°C as cell extract.
  • Monoclonal antibodies LJ-Ibl Handa et al. and LJ-P19, which recognize GPIb ⁇ native conformation were used as primary antibody.
  • the secondary antibody 125 I-rabbit anti-mouse IgG which had been labelled by the method of Fraker et al. , Biochem. Biophys. Res. Commun. , 80, 849-857 (1978) was incubated for 2 hours at 25°C on a nitrocellulose sheet. After rinsing, the nitrocellulose was developed by autoradiography to identify colonies expressing GPIb ⁇ antigen.
  • Extracts from pMWl-transformed cells contain as a minor component a glycoprotein Ib ⁇ antigen having an approximate apparent molecular weight of 79 kDa as measured by SDS- - polyacrylamide gel electrophoresis under reducing or non- reducing conditions.
  • This band represents full length glycoprotein Ib ⁇ chain (residues 1-610 and minus the signal peptide) without glycosylation.
  • the 79 kDa polypeptide reacts with anti-GPIb ⁇ monoclonal antibody LJ-IB ⁇ l which has its epitope in the amino terminal region of the denatured ( ⁇ ) polypeptide, whether in reduced or unreduced form. The relatively small proportion of this species indicates its inherent instability and rapid proteolytic processing.
  • the 79 kDa polypeptide was not detected in culture medium from pMWl transformed cells. Instead the major GPIb( ⁇ ) polypeptide isolated from such medium has an approximate apparent molecular weight of 45 kDa, characteristic of the properly glycosylated amino terminal domain of GPIb ⁇ .
  • the presence of this species in the culture medium of pMWl transformed cells demonstrates that the amino terminal domain of GPIb ⁇ can be processed as a secretory protein and reaches structural maturation (A) in the absence of assembly of the other components of the GPIb complex and (B) in spite of the usual proteolysis of the full length GPIb ⁇ polypeptide.
  • the His ⁇ Ala 302 fragment contains sufficient primary sequence information to be assembled into a structure possessing domains of tertiary structure present in native glycoprotein Ib ⁇ .
  • a functionally equivalent His ⁇ Arg 293 receptor fragment for vWF or thrombins
  • oligonucleotides were synthesized by the phosphoramidite method, Sinha et al., using a model 380B automated system. Applied Biosystems, Foster City, CA. Nucleotides are shown using the numbering system of Wenger et al. for the GPIb ⁇ gene.
  • Oligonucleotide (D) (SEQ ID NO: 14)
  • Oligonucleotide (C) is equivalent to nontranscribed (coding) strand DNA.
  • Oligonucleotide (D) shown in capital letters, is equivalent to transcribed (noncoding) strand DNA.
  • the corresponding coding strand for oligonucleotide (D) is shown 5' ⁇ 3' with the encoded amino acids shown by standard three letter designation.
  • a BamHl linker was added to the amplified double stranded DNA sequence 3' to the partial Ala 302 codon thereby completing the codon and enabling the DNA to function as a BamHl insert. Roberts et al.. Nature. 265, 82-84 (1977).
  • the amplified fragment was then cloned into the BamHl site within the multiple cloning sequence of the double stranded replicative form of M13mpl9 bacteriophage.
  • the ability to isolate a stable single stranded (+) form of the virus is particularly useful to verify the integrity of any cloned sequences therein. See, for example, Messing and Yanish-Perron et al.
  • the GPIb ⁇ DNA insert was completely sequenced using single stranded dideoxy methodology, Sanger et al., utilizing the single stranded (+) form of M13mpl9 to confirm that the GPIb ⁇ fragment contained the correct coding sequence for the region of GPIb ⁇ DNA represented by nucleotides 502 to 1489 and including a codon for the initiating methionine, the remaining 15 residues of the signal peptide and residues 1 to 302 of the amino terminal region of mature GPIb ⁇ polypeptide.
  • E. coli strain SX-127 cells Ampicillin sensitive E. coli strain SX-127 cells (Invitrogen, San Diego, CA) were transformed with the resultant ligated DNA mixture following the method of Hanahan, J. Mol. Biol.. 166, 557-580 (1983). Plasmids from resultant colonies were characterized by restriction mapping and DNA sequencing to identify colonies which contained the intended insert. One such appropriate plasmid (designated pMW2) was maintained in E. coli strain XS-127, and was selected for mammalian cell transformation procedures.
  • supercoiled plasmids Prior to use in transforming mammalian cells, supercoiled plasmids (pMW2) were recovered from host E. coli by the alkaline lysis procedure of Birnboim and Doly followed by CsCl/ethidium bromide equilibrium centrifugation according to the procedure described above. Transformation of CH0-K1 cells also followed the procedure therefor described above for pMWl plasmid.
  • Example 3 Expression of a Mutant Cysteine-Free Mature von Willebrand Factor Subunit Fragment Having an Amino Terminus at Residue 441 (Arginine) and a Carboxy Terminus at Residue 733 (Valine) Preparation of a cDNA Clone from pre-pro-von Willebrand Factor mRNA
  • Lynch Dana-Farber Cancer Institute, Boston, MA and was. prepared as described in Lynch, D.C. et al. , Cell, 41, 49-56
  • RNA from endothelial cells (the major source of plasma vWF) was sedimented in sucrose gradients, with RNA larger than 28S being selected for construction of a cDNA library.
  • This enriched fraction was further purified using two separate cycles of poly(u)-Sephadex ® chromatography to select for RNA species (mRNA) having 3' polyadenylated ends.
  • Lynch et al., supra estimated the prevalence of vWF mRNA in this fraction at about 1 in 500, which fraction was used to generate a cDNA library of approximately 60,000 independent recombinants.
  • RNA population was primed using an oligo (dT) primer, and then transcribed with a reverse transcriptase.
  • dT oligo
  • RNA strands were then removed by alkaline hydrolysis, leaving cDNA anticoding strands (equivalent to transcribed strands) which were primed by hairpin looping for second strand synthesis using DNA polymerase I.
  • the hairpin loop was removed with Sj nuclease and rough ends were repaired with DNA polymerase I.
  • GC tailing Maniatis, T. et al., Molecular Cloning, 2n ed., v.l, p.5.56 (1987), was then used to anneal the cDNA into plasmid vector pBR322. Oligo(dC) tails were added to the cDNA fragments with terminal transferase and were annealed to oligo(dG) tailed pBR322. The plasmids were transformed into ampicillin sensitive E.coli. strain HB101 for propagation. Suitable clones were identified after screening with 32 P-labelled cDNA prepared as reverse transcriptase product of immunopurified vWF polysomes.
  • oligonucleotides flanking the region of interest (designated #440, SEQ ID NO: 15, and #730, SEQ ID NO: 16) were prepared. All oligonucleotides used herein were synthesized by the phosphoramidite method , Sinha, et al., Tetrahedron Letters, 24, 5843 (1983), using a model 380B automated system, Applied Biosyste s, Foster City, CA. Oligonucleotide #440 (SEQ ID NO: 15)
  • Oligonucleotides overlap the ends of the coding region for that fragment of the mature vWF subunit which can be produced by digestion with trypsin and which begins with residue 449 (valine) and ends with residue 728 (lysine) .
  • Oligonucleotide #440 corresponds to coding strand DNA (analogous with mRNA) for amino acid positions 441 to 446 and adds an EcoRI restriction site 5' to the codon for amino acid 441.
  • Oligonucleotide 730 corresponds to the non-coding strand (transcribed strand) of mature vWF DNA for amino acids positions 725-733 and adds a Hindlll restriction site 3' to the codon for amino acid 733.
  • the coding strand complementary to #730 is shown in lower case letters.
  • a cDNA fragment corresponding to mature vWF residues Nos. 441-733, and containing EcoRI and Hind III linkers was then synthesized in a polymerase chain reaction following the method of Saiki, R.K. et al. Science, 239, 487- 491 (1988) .
  • the procedure utilizes a segment of double-stranded vWF cDNA, a subsegment of which is to be amplified, and two single-stranded oligonucleotide primers (in this case oligonucleotides #440, 730) which flank the ends of the subsegment.
  • the primer oligonucleotides in the presence of a DNA polymerase and deoxyribonucleotide triphosphates were added in much higher concentrations than the DNA to be amplified.
  • cDNA corresponding to the mature vWF fragment beginning at amino acid sequence position 441 and ending at position 733 was prepared and amplified directly from platelet mRNA following the procedure of Newman, P.J. et al. J. Clin. Invest. , 82, 739-743 (1988). Primer nucleotides No. 440 and 733 were utilized as before with the resulting cDNA containing EcoRI and Hindlll linkers. Insertion of cDNA into M13mpl8 Cloning Vehicle
  • M13 series filamentous phages infect male (F factor containing) E.coli strains.
  • the infecting form of the virus is represented by single stranded DNA, the ( + ) strand, which is converted by host enzymes into a double stranded circular form, containing also the minus ( " ) strand, which double stranded structure is referred to as the replicative form (RF) .
  • RF replicative form
  • vWF cDNA insert was completely sequenced using single-stranded dideoxy methodology (Sanger, F. et al. Proc. Natl. Acad. Sci USA. 74, 5463-5467 (1977)), utilizing the single-stranded ( + ) form of M13mpl8, to confirm that the vWF cDNA fragment contained the correct coding sequence for mature vWF subunit residues 441-733.
  • oligonucleotides see Sequence Listing ID NOS: 17-20) encompassing the region of each cysteine codon of the vWF cDNA were prepared as non-coding strand (transcribed strand) with the corresponding -base substitutions needed to substitute glycine for cysteine.
  • the oligonucleotides used were as follows: Oligonucleotide #459 (SEQ ID NO: 17)
  • Oligonucleotide #471 (SEQ ID NO: 18)
  • Oligonucleotide #695 (SEQ ID NO: 20) 3'TCG ATG GAG CCA CTG GAA CGG5'
  • Hybridizing oligonucleotides are shown in capital letters and are equivalent to the transcribed strand (non ⁇ coding DNA) .
  • the equivalent coding strand is shown in lower case letters with the corresponding amino acids shown by standard three letter designation, (for designations see Table 1)
  • cysteines 459, 462 and 464 were replaced simultaneously using oligonucleotide 459. Cysteine residues 471 and 474 were then replaced simultaneously using oligonucleotide 471. Cysteine residues 509 and 695 were then replaced individually using oligonucleotides 509 and 695 respectively.
  • cysteine to glycine cDNA substitutions were accomplished following the procedure of Kunkel, T.A. , Proc. Natl. Acad. Sci. USA. 82,488-492 (1985) which procedure repeats a series of steps for each oligonucleotide and takes advantage of conditions which select against a uracil containing DNA template:
  • Viral DNA synthesized under these conditions includes several uracil insertions per viral genome and is stable only in an E.coli strain which is incapable of removing uracil, such as ( ng ⁇ ) strains which lack uracil glycosylase.
  • Uracil-containing nucleotides are lethal in single strande ( + ) M13mpl8 DNA in ung + strains due to the creation of abasic sites by uracil glycosylase.
  • Oligonucleotide #459 which contains codon alterations necessary to substitute glycines for cysteines at positions 459, 462 and 464, is then annealed in vitro to single stranded ( + ) phage DNA.
  • oligonucleotide concentrations is suitable in this procedure.
  • ng of oligonucleotide was annealed to 0.5-1.0 ⁇ g M13mpl8 phage ( + ) DNA.
  • the double stranded vWF cDNA fragment containing 7 sit specific cysteine to glycine mutations is then removed from M13mpl8 phage by treatment with EcoRI and Hindlll restricti endonucleases, after which the ends of the fragment are modified with BamHl linkers (Roberts, R.J. et al. Nature. 265, 82-84 (1977)) for cloning into a high efficiency E.col expression vector.
  • the particular expression vector chosen is plasmid pET-3A, developed by Rosenberg, A.H. et al.
  • the pET-3A vehicle is refered to as "p7E” or p7E expression plasmid.
  • a second pET-3A-derived expression plasmid (designated p7D) was constructed containing the identical vWF coding sequence cloned into the plasmid in the opposite orientatio p7D should be unable to express the vWF polypeptide fragmen
  • a third expression plasmid (pJD18) contains wild type
  • the p7E (or p7D and pJD18) expression plasmids were the cloned into an ampicillin sensitive E.coli strain, BL21(DE3) Novagen Co., Madison WI, according to a well established protocol Hanahan, D., J. Mol. Biol.. 166, 557-580 (1983).
  • Strain BL21(DE3) is engineered to contain a gene for T7 RNA polymerase so that the vWF insert can be transcribed with high efficiency.
  • E.coli strain BL21(DE3) containing respectively p7E, p7D or pJD18 expression plasmid were innoculated into 5-6 ml of 2X-YT growth medium containing 200 ⁇ g/ml of ampicillin, and grown overnight at 37°C to create fully grown cultures.
  • 2X-YT growth medium contains, per liter of water, 10 g Bacto-tryptone, 10 gm yeast extract and 5 gm NaCl. Five ml of each overnight culture was then innoculated into 500 ml of 2X-YT medium, again containing 200 ⁇ g/ml of ampicillin and grown for 2 hours at 37°C with shaking.
  • vWF polypeptide A high level of expression of vWF polypeptide was obtained with p7E and pJD18 resulting in the generation of cytoplasmic granules or "inclusion bodies" which contain high concentrations of vWF polypeptide in essentially insoluble form. Solubilization of vWF polypeptide was accomplished according to the following procedure. As explained in Example 4, p7E and pJDl ⁇ extracts responded very differently to solubilization procedures. See Maniatis, T. et al., Molecular Cloning. 2nd ed. , vol. 3, Sec. 17.37, (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, for a general discussion of the properties of, and successful manipulation strategies for, inclusion bodies.
  • the cells were harvested by centrifugation at 4000 g for 15 minutes in a JA-14 rotor at 4°C.
  • the pelleted cells were washed in 50 ml of ice cold buffer (0.1 M NaCl, 10 mM Tris pH 9.0, 1 mM EDTA) and repelleted by centrifugation at 4000 g at 4°C.
  • the cell pellets from p7E, p7D and pJD18 cultures were ⁇ each redissolved in 5 ml of lysing buffer and kept ice-cold for 30 minutes.
  • the lysing buffer comprises a solution of sucrose 25%(w/v) , 1 mM phenylmethylsulfonylfluoride (PMSF) , 1 mM ethylene dia inetetraacetic acid (EDTA) , 2 mg/ml lysozyme and 50 mM Tris hydrochloride, adjusted to pH 8.0.
  • the relatively insoluble pelleted material derived from each culture (which contains the desired polypeptides except in the case of p7D) was washed at 25°C in 10 ml of buffer No. 2 (0.5% (w/v) Triton X-100 surfactant, 2 mM EDTA, 0.02 M Tris hydrochloride, pH 7.5) and vortexed extensively. The suspension was centrifuged at 14,000 g for 30 minutes at 4°C and the supernatant was then discarded. The process of resuspension of the pelleted material in buffer No. 2, vortexing and centrifugation was repeated twice.
  • each pellet was then washed in 5 ml of buffer No. 3 (0.02 M Tris hydrochloride, pH 7.5, and 2 mM EDTA) at 25°C and vortexed extensively. The suspension was then centrifuged at 4°C for 30 minutes at 14,000 g after which the supernatant was discarded leaving a pellet of inclusion body derived material with a clay-like consistency.
  • buffer No. 3 0.2 M Tris hydrochloride, pH 7.5, and 2 mM EDTA
  • the insoluble pellet was slowly redissolved in an 8 Molar urea solution held at room temperature for 2 hours, after which solubilization was continued overnight at 4°C.
  • the urea-soluble material was extensively dialyzed against a solution of 0.15 M NaCl containing 20 mM Hepes (N-[2- hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]) (pH 7.4) at 4°C.
  • the solublized peptide extracts were assayed for purity (Example 4) , used in vWF binding inhibition assays, or subject to further purification. Further purification steps should not be delayed and the samples should remain cold.
  • the cysteine-free vWF polypeptide (comprising subunit positions 441 to 733) constitutes more than 75% of the material solubilized from the inclusion bodies according to the above procedure. Further purification of the cysteine- free mutant vWF polypeptide is accomplished by redialyzing the partially purified peptide extract against 6 M guanidine-HCl, 50 mM Tris-HCl, pH 8.8 followed by dialysis against 6 M urea, 25 mM Tris-HCl, 20 mM KC1, 0.1 mM EDTA, pH 8.0. The extract is then subjected to chromatography on a Q- Sepharose ® column (Pharmacia, Uppsala, Sweden) .
  • the column was preequilibrated with 6 M urea, 25 mM Tris*HCl, 20 mM KC1, 0.1 mM EDTA pH 8.0. Elution of the vWF polypeptide utilized the same buffer except that the concentration of KCl was raised to 250 mM. Polypeptide samples used for further assays were redialyzed against 0.15 M NaCl, 20 mM Hepes, pH 7.4. However, long term storage is best achieved in urea buffer (6 M urea, 25 mM Tris-HCl, 20 mM KCl, 0.1 mM EDTA pH
  • Example 4 Characterization of the Cysteine-Free Mutant von Willebrand Factor Fragment Produced by Expression Plasmid p7E Urea-solubilized and dialyzed polypeptides extracted from inclusion bodies of cultures containing expression plasmids p7E, p7D and pJD18 were analyzed using polyacrylamide gel electrophoresis (PAGE) and immunoblotting.
  • PAGE polyacrylamide gel electrophoresis
  • the resultant gels were stained with Coomassie blue and compared.
  • the extract from expression plasmid p7E contains as the major component, the mutant von Willebrand factor polypeptide which migrates with an apparent molecular weight of approximately 36,000 Daltons.
  • the polypeptide appears as a single band under both reducing conditions (addition of between 10 and 100 mM dithiothreitol "DTT" to the sample for
  • vWF polypeptide could be extracted from host cells containing p7D expression plasmids as expected from the opposite orientation of the vWF cDNA insert.
  • the wild-type sequence expressed from pJD18 forms intermolecular disulfide bridges resulting in large molecular weight aggregates which are unable to enter the 10% acrylamide gels. After reduction (incubation with 100 mM DTT for 5 min at 100°C) , the vWF peptide migrates as a single band with a molecular weight of approximately 38,000.
  • Example 5 Expression of a Mutant vWF Fragment of Reduced Cysteme Content Containing a
  • the double-stranded form of the vWF cDNA insert (containing 5 cysteine to glycine mutations) was then removed from M13mpl8 phage by treatment with EcoRI and Hindlll restruction endonucleases, modified as in Example 3 with BamHl linkers, and cloned into pET-3A.
  • the pET-3A vehicle so formed is referred to as "p5E” or p5E expression plasmid.
  • the p5E expression plasmids were then cloned into ampicillin sensitive E.coli strain BL21(DE3), Novagen Co., Madison, WI, according to the procedure of Hanahan, D., J ⁇ . Mol. Biol.. 166, 557-580 (1983).
  • the p5E mutant polypeptide was expressed from cultures of E.coli BL21(DE3) following th procedure of Example 3 except that solubilization of inclusion body pellet material in the presence of 8 Molar urea need not be continued beyond the initial 2 hour period at room temperature, at which point redissolved material had reached a concentration of 200 ⁇ g/ml.
  • Example 3 As in Example 3 pertaining to the p7E extracts, final purification of urea-solubilized inclusion body preparations was accomplished by dialysis against 6 M guanidine and urea solutions followed by Q-Sepharose ® chromatography.
  • vWF Fragment Useful as a Domain of the Bifunctional Molecules of the Invention This example is illustrative of the preparation of domains of bifunctional antithrombotic molecules that are derived from the residue 441-733 fragment of vWF subunit. The example is also illustrative of conditions under which such vWF subfragments may be expressed from recombinant bacterial host cells. The subfragments may be expressed also from recombinant eucaryotic cells, for example, by following the general procedures of Example 7.
  • the vWF subfragments are capable of interfering with the interaction of multimeric vWF and platelet GPIb ⁇ , that is, they have utility as a domain of a bifunctional antithrombotic molecule.
  • p7E plasmids were recovered from cultures of E.coli BL21(DE3) using an alkaline cell lysis procedure,
  • Oligonucleotide (E) see SEQ ID NO: 21 3' - GAG TGG CCA CTT CGG CAC TCG GGG TGG TGA - 5'
  • Oligonucleotide (F) - see SEQ ID NO: 22
  • the resultant mutant (fusion) vWF polypeptides were then tested for their ability to bind to GPIb ⁇ . Using an assay dependent on inhibition of the binding of LJ-Ibl antibody to GPIb ⁇ in the absence of botrocetin modulator, it was determined that the residue 441-733 fragment, which was expressed from p7E and from which the "G10" peptide sequence was deleted, binds GPIb ⁇ . The p7E-derived fusion fragment lacking the "D5" peptide sequence did not. However, when the experiments were repeated using botrocetin as a modulator of binding, both of the fused subfragments were effective in inhibiting binding by LJ-Ibl, and hence have utility as domains of bifunctional antithrombotics.
  • vWF-derived domains having such antithrombotic activity include inhibition of botrocetin-induced binding of vWF to platelets by the vWF-derived domain, and the inhibition of human platelet agglutination in a system using bovine vWF, but without a modulator such as botrocetin or ristocetin.
  • Polypeptide subfragments effective as domains of bifunctional antithrombotics have also been prepared which are patterned upon the residue 441-733 vWF subunit fragment, but which contain N-terminal deletions therefrom.
  • p7E sequence is also useful for expression of such antithrombotic domains.
  • Antithrombotic polypeptide domains equivalent to those expressed from p7E constructs can be made by reduction and alkylation of cysteine residues otherwise contained therein.
  • oligonucleotides used to create N-terminal deletions in the vWF subunit fragment made reference to DNA sequence of the pET-3A vector that is upstream (5') from the codon encoding vWF residue 441. Expression of the residue
  • 3A involves expression also of a twenty residue amino acid sequence (SEQ ID NO: 23) that remains attached to the amino terminal of the vWF fragment.
  • This sequence as shown below, is encoded by vector DNA downstream from the T7 promoter site but does not affect adversely the therapeutic activity of the vWF polypeptide. initiation codon i
  • Oligonucleotide (G) - see SEQ ID NO: 24
  • oligonucleotide H SEQ ID NO: 25
  • Shown below this oligonucleotide are the corresponding coding strand and resultant amino acids.
  • vWF subfragments reflecting such N- terminal deletions are Met- Gin 475 to Val 733 (SEQ ID NO: 26), Met-Thr 492 to Val 733 , and Met- Tyr 508 to Val 733 .
  • Such vWF subfragments (and other species having terminal deletion of any subsets of the vWF residue 441-508 sequence) have anti- thrombotic therapeutic activity.
  • These polypeptides can present also the cysteine 509-695 loop when expressed from p5E constructs.
  • vWF Subfragments Having C-Terminal Deletions The procedure used.to express recombinant bacterial polypeptides using pET-3A vectors results in polypeptides that comprise also a series of amino acids on the C-terminal side of Val 733 , the additional residues arising from translation of vector sequence (see SEQ ID NO: 27 below) .
  • residue 441-733 fragments expressed from p5E (or p7E) constructs contain also 22 residues fused to the C-terminal side of residue 733 (valine) resulting from the expression of vector sequence prior to the first vector stop codon.
  • This additional vector sequence does not affect adversely the therapeutic utility of the resultant polypeptide.
  • other DNA expression constructs targeting for either the added amino or carboxy terminal vector sequences could be designed to avoid the additional sequences.
  • This pET-3A vector sequence which reflects also modification (Example 3) of the Hindlll site of the EcoRI- Hindlll fragment by a BamHl linker, is (SEQ ID NO: 27) :
  • loopout mutagenesis was performed in p5E using hybridizing oligonucleotides patterned on non-coding strand DNA.
  • a hybridizing oligonucleotide was created encoding vWF subunit sequence (for example, from residue 706 to 713) that included also between certain codons thereof (for example, codon 709 and codon 710) the stop codon/reading frame shift sequence 3' - ACT-ACT-T - 5'.
  • vWF-derived polypeptide subfragments useful in the practice of the invention were generated that have c- terminal deletions and which terminate at residues 709 (SEQ ID NO: 28) , 704, 700 and 696 respectively.
  • Fragments of von Willebrand factor subunit useful as domains of the bifunctional antithrombotic molecules of the invention may be prepared also from recombinant euearyotie host cells.
  • vWF-derived polypeptides can be expressed in such host cells, or secreted therefrom if appropriate DNA constructs are used containing signal peptide encoding sequence.
  • Procedures necessary for the expression of the Arg 441 to Asn 730 fragment of mature vWF subunit from mammalian host cells are described in the publication Azuma, H. et al., Independent Assembly and Secretion of a Dimeric Adhesive Domain of von Willebrand Factor Containing the Glycoprotein lb-Binding Site, J. Biol.
  • the hybridizing oligonucleotide is shown (3' ⁇ 5') in capital letters and is equivalent to transcribed strand (non ⁇ coding strand DNA) . Underlined letters indicate the single base mutations for the mutant codons. The equivalent coding strand is shown in lower case letters with the corresponding glycine substitutions identified by three letter designation.
  • Example 8 Determination of the variable region primary amino acid sequences of LJ-Ibl0
  • antibody molecules are tetrameric immunoglobulins consisting of two identical light (L) and two identical heavy (H) polypeptide chains.
  • the N-terminal region of each chain designated V L or V H , is usually 110 amino acid residues long and is composed of 3 hypervariable or complementarity-determining regions (CDR) and 4 less variable or framework (FR) regions.
  • CDR complementarity-determining regions
  • FR variable or framework
  • the N-terminal region of the light and heavy chain variable regions usually consists of four FR sequences interspersed by 3 CDR sequences, FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4 (in amino to carboxyl order) .
  • the conformation of the antibody combining sites is determined by the amino acid residues of the CDR (Amit, A.G. et al., Science, 233, 747-758 (19 ⁇ 6) ) .
  • the CDR sequences provide the unique characteristic features of the antibody, whereas the FR regions provide the structural scaffolding that aligns the CDR.
  • the cells are then resuspended in a lysis buffer composed of 4 M guanidine isothiocyanate, 20 mM sodium acetate (pH 5.2), 0.1 mM dithiothreitol, and 0.5% n-lauryl sarcosine.
  • a lysis buffer composed of 4 M guanidine isothiocyanate, 20 mM sodium acetate (pH 5.2), 0.1 mM dithiothreitol, and 0.5% n-lauryl sarcosine.
  • the lysate is passed 3-4 times through a syringe containing a 20 gauge needle to reduce the viscosity of the lysate.
  • the sheared lysate is then layered on top of a 5.7 M cesium chloride cushion and centrifuged for 20 hours at approximately 100,000 x g.
  • the principle of the procedure see Glisin, V. et al. , Biochemistry, 13, 2633
  • the total RNA can then be subjected to a first strand cDNA reaction using Moloney murine leukemia virus reverse transcriptase using the recommended assay conditions for the enzyme.
  • the first strand reaction is then primed with oligonucleotides designated VHIFOR (specific for FR4 of heavy chain) and VKIFOR ( ⁇ -chain variable primer analogous to FR4 of the light chain) shown below:
  • oligonucleotides were designed to represent non ⁇ coding strand sequence for the indicated regions but are not necessarily an exact match for the noncoding strand of all light and heavy chain genes.
  • the oligonucleotides along with others suitable for generating an antibody cDNA have been described by Orlandi, R. et al., Proc. Natl. Acad. Sci. USA, 86, 3844-3847 (1989); Chiang, Y.L. et al., BioTechnigues, 7, 360-366 (1989); Larrick, J.W. et al., Bio/Technology, 7, 934- 938 (1989); Loh, E.Y. et al.. Science.
  • oligonucleotides represent noncoding strand sequence and will anneal to the corresponding coding sequence present within the specific mRNA species responsible for the generation of LJ-IblO.
  • the amplified fragments can then be cloned into M13 vectors as Pstl/Kpnl fragments (heavy chain) and Pstl/Bglll fragments (light chain) .
  • the nucleotide sequence of the inserts would be determined by DNA sequence analysis. It is known that the hybridoma cells secreting LJ-IblO also secrete an antibody designated MOPC-21.
  • MOPC-21 The myeloma cell line used in the initial fusion to generate the LJ-IblO-secreting hybridoma secretes the MOPC-21 immunoglobulin.
  • the sequence of MOPC-21 is known (Kabat, E.A. et al.. Sequences of Proteins of Immunological Interest. U.S. Publick Health
  • a F jb fragment can be prepared easily by proteolytic cleavage of an antibody.
  • F Ib fragments have the same antigen binding capacity as the whole antibody.
  • Generating a F ⁇ fragment is well exploited technique to reduce the size of an immunoglobulin and to avoid numerous possible complications (see above) associated with expression of a full size antibody construct.
  • only half of the F, b fragment is required for antigen binding because it is composed of a constant region (approximately one-half of the F ⁇ ) and two variable region binding arms.
  • An F v fragment represents generally the smallest antibody fragment that still contains an antigen binding site. It is essentially composed of the 4 framework regions interspersed with the 3 CDR for both a heavy and a light chain in combination.
  • F v fragments are difficult to prepare by proteolysis, but have become the structural framework for synthesizing recombinant antibodies using genetic techniques.
  • the proposed strategy there is first generated a single-chain F v polypeptide sequence (consisting of the light chain variable region only), a linker polypeptide sequence, and then the heavy chain variable region.
  • Site-directed mutagenesis strategies can be used to fuse the cDNA coding sequences for each region is a single open reading frame that will produce the F v in the form of single-chain polypeptide.
  • An M13 construct containing the coding sequence for heavy chain variable region (as a KpnI-PstI fragment) can be mutated so that it is flanked by the recognition sequence for the restriction enzyme BamHl.
  • the mutagenesis procedure would use established technology as described by Kunkel, T.A. et al.. Methods in Enzymology. 154, 367-383 (1987) , see also Example 3 herein.
  • the Pstl-BamHI fragment containing the coding sequence for the light chain variable region would be excised from the M13 construct and cloned as a Pstl-BamHI fragment into pBS/KS- (Stratagene, La Jolla, CA) . After isolation of recombinant plasmids containing the light chain cDNA, the BamHl fragment containing the heavy chain sequence can be cloned into the recombinant plasmid containing the light chain sequence. A representative recombinant.
  • plasmid would then be identified through restriction enzyme analysis and DNA sequence analysis that contains the light-heavy chain cDNA fragment oriented in a manner that would, in a 5' ⁇ 3' coding direction, contain coding sequence for light chain variable sequence followed by coding sequence for heavy chain variable sequence.
  • linker or "spacer” sequence generating the single open reading frame for the light and heavy chain polypeptides separated by a polypeptide sequence that facilitates the in vitro association of light and heavy chains.
  • linkers have been described (Huston, J.S. et al. , Proc. Natl. Acad. Sci. USA. 85, 5879-5883 (1988);
  • Ser 3 .
  • the pET3a vector allows the isolation of single-chain F v fragments from within inclusion bodies, and their subsequent purification uses established procedures (see Example 3) .
  • Characterizations of a recombinant LJ-IblO F v could include: (1) reactivity with GPIb ⁇ antigen either on the platelet surface or using a recombinant antigen that contains the epitope of LJ-IblO (Murata, M. et al., J. Biol. Chem.9, 266, 15474-15480 f!99in or (2) the ability of the recombinant LJ-IblO F v polypeptide to inhibit the binding of
  • a bifunctional inhibitor molecule of vWF and ⁇ -thrombin binding to platelet GPIb ⁇ There can be prepared a chimeric cDNA fragment that comprises coding sequence for a vWF fragment that contains the. binding site thereof for GPIb ⁇ fused to the coding sequence for the "single-chain" LJ-IblO F v described above.
  • the cloning strategy used herein is similar to that used to create the single-chain light and heavy chain fusion construct of LJ-IblO.
  • each fragment can be modified to generate the following elements in a 5' to 3' coding direction: sequence for a vWF fragment that contains the GPIb binding site, as example residues 441-728; a "linker” or “spacer” composed of Gly codons to insure flexibility within the linker polypeptide; the coding sequence for the F v LJ-IblO fragment.
  • the mutagenesis can be performed on the coding sequence of LJ-IblO F v within the pBS/KS- construct described above.
  • the mutagenesis for the vWF fragment can be performed on constructs, such as that previously described (see Example 3).
  • Each fragment can be altered to contain a restriction recognition sequence for an enzyme recognizing an eight-base DNA sequence, such as NotI, Sfil and Pad.
  • the eight base sequence will occur once every 65,536 nucleotides (1/4 8 ) and these restriction enzymes are commercially available from New England Bio-Labs (Beverly, MA) .
  • the probability that two fragments ranging in size from 600-900 base pairs would contain the restriction recognition sequence of all three enzyme sequences is extremely low.
  • Other mutagenesis strategies could involve the elimination of recognition sequences within the fragments without altering the translated amino acid sequence (taking advantage of the degenerate nature of the genetic code) to allow a ligation of the coding sequences.
  • the final chimeric cDNA fragment will be cloned into pET3a for the preparation of inclusion bodies (see, for example. Example 3) and subsequent isolation of the bifunctional recombinant fragment.
  • a chimeric molecule consisting of an inhibitor of plasma vWF binding to GPIb ⁇ and for binding of ⁇ -thrombin to GPIb ⁇
  • This molecule will be composed of the polypeptide that mimics the GPIb binding site within the vWF (a vWF polypeptide fragment) fused to coding sequence for GPIb ⁇ residues 271-285.
  • the vWF fragment construct will be subjected to site-directed mutagenesis (Example 3) to add the indicated GPIb ⁇ coding sequence.
  • An oligonucleotide can then be synthesized that contains GPIb ⁇ coding sequence for residues 271-285 flanked by sequence present in an M13 construct that contains the coding sequence for a vWF fragment 441-728.
  • the mutagenesis strategy can be employed to insert the GPIb ⁇ peptide sequence at the carboxyl-terminal region of the vWF fragment although insertion at the amino terminus is equally possible.
  • An additional "linker" peptide sequence will be included to prevent any structural constraint that might be imposed by the vWF fragment on the
  • GPIb ⁇ sequence or vice-versa.
  • Murine hybridoma LJ-IblO ATCC HB10939 12/3/91 (TSRI 138.5)
  • Murine hybridoma LJ-Ibl ATCC HB10940 12/3/91 (TSRI 138.5)

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Abstract

Une molécule bifonctionnelle peut inhiber l'activation d'une plaquette et/ou l'adhésion d'une plaquette sur un domaine vasculaire endommagé ou malade, ladite molécule bifonctionnelle comprenant deux domaines liés, un premier domaine pouvant empêcher la liaison de thrombines sur la glycoprotéine Ibalpha des plaquettes et un second domaine pouvant empêcher la liaison du facteur von Willebrand sur la glycoprotéine Ibalpha des plaquettes. L'invention décrit également une composition thérapeutique contenant la molécule bifonctionnelle. Le procédé de production de la molécule bifonctionnelle consiste à cultiver un organisme hôte transformé avec un plasmide d'expression biologiquement fonctionnel qui contient une séquence ADN codant la molécule bifonctionnelle dans des conditions qui permettent d'effectuer l'expression de la molécule par l'organisme hôte, et de récupérer la molécule de l'organisme hôte.
PCT/US1992/010947 1991-12-12 1992-12-11 Molecules anti-thrombotiques bifonctionnelles et polypetptides anti-trhombotiques WO1993011778A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0627923A4 (fr) * 1992-02-26 1996-01-17 Scripps Research Inst Domaines therapeutiques du facteur von willebrand.
US7132510B2 (en) 2000-12-29 2006-11-07 Bio-Technology General (Israel) Ltd. Specific human antibodies for selective cancer therapy
EP1406930A4 (fr) * 2000-12-29 2007-01-10 Savient Pharmaceuticals Inc Molecules isolees renfermant des epitopes a fractions sulfatees, anticorps vis-a-vis de ces epitopes, et utilisations correspondantes
WO2008006189A1 (fr) * 2006-07-11 2008-01-17 Canadian Blood Services Récepteurs et inhibiteurs mimotopes pour les interactions plaquettes-plaquettes et plaquettes-endothelium
WO2018148455A1 (fr) * 2017-02-08 2018-08-16 HKL Medical, LLC Procédés d'administration et de dosage intrasite et produits pharmaceutiques destinés à y être utilisés

Citations (1)

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Publication number Priority date Publication date Assignee Title
US4816397A (en) * 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US4816397A (en) * 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production

Non-Patent Citations (4)

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Title
BLOOD, Volume 76, No. 5, issued 01 September 1990, HORTIN, "Sulfation of Tyrosine Residues in Coagulation Factor V", pages 946-952. *
J. BIOL. CHEM., Volume 261, No. 27, issued 25 September 1986, HANDA et al., "The Von Willebrand Factor-Binding Domain of Platelet Membrane Glycoprotein Ib", pages 12579-12585. *
J. BIOL. CHEM., Volume 264, No. 29, issued 15 October 1989, MOHRI et al., "Isolation of the Von Willebrand Factor Domain Interacting with Platelet Glycoprotein Ib, Heparin, and Collagen and Characterization of its Three Distinct Functional Sites", pages 17361-17367. *
J. CLIN. INVEST., Volume 86, issued July 1990, DE MARCO et al., "Variant Bernard-Soulier Syndrome Type Bolzano - A Congenital Bleeding Disorder Due to a Structural and Functional Abnormality of the Platelet Glycoprotein Ib-IX Complex", pages 25-31. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0627923A4 (fr) * 1992-02-26 1996-01-17 Scripps Research Inst Domaines therapeutiques du facteur von willebrand.
US7132510B2 (en) 2000-12-29 2006-11-07 Bio-Technology General (Israel) Ltd. Specific human antibodies for selective cancer therapy
EP1406930A4 (fr) * 2000-12-29 2007-01-10 Savient Pharmaceuticals Inc Molecules isolees renfermant des epitopes a fractions sulfatees, anticorps vis-a-vis de ces epitopes, et utilisations correspondantes
WO2008006189A1 (fr) * 2006-07-11 2008-01-17 Canadian Blood Services Récepteurs et inhibiteurs mimotopes pour les interactions plaquettes-plaquettes et plaquettes-endothelium
US7919465B2 (en) 2006-07-11 2011-04-05 Maria I. C. Gyongyossy-Issa Peptide mimotopes that inhibits interaction between a platelet receptor and a platelet receptor ligand
WO2018148455A1 (fr) * 2017-02-08 2018-08-16 HKL Medical, LLC Procédés d'administration et de dosage intrasite et produits pharmaceutiques destinés à y être utilisés

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